Methods of screening for pharmacologically active compounds for the treatment of tumour diseases

ABSTRACT

Pharmaceutical compositions containing as active compound a substance which inhibits the activity of TGFβ on tumour cells of epithelial origin, for the treatment of epithelial, invasive tumour diseases which are characterized by a reversible transition of the cells from an epithelial, non-invasive state into a fibroblastoid, invasive state. The pharmaceutical composition contains a TGFβ inhibitor, preferably combined with an Ras inhibitor. Process for screening substances for the treatment of epithelial, invasive tumour diseases.

FIELD OF THE INVENTION

The invention relates to the field of tumour therapy.

BACKGROUND OF THE INVENTION

More than 80% of tumours occurring in man are of epithelial origin. Theformation of epithelial tumours (carcinomas) is a multi-stage processwhich is illustrated most clearly in the progression of human coloncarcinoma (Powell, et al., 1993) and skin tumours in mice (Wright, etal., 1994). Carcinomas are assumed to start from individual cells orsmall groups of cells in which mutations have occurred. These cellsdevelop into benign, epithelial hyper- or dysplastic regions. Theprogression of these hyperplastic regions into a carcinoma in situ,which may then acquire invasive and metastatic properties, requires anumber of further mutations in the tumour cell. Characteristically thesecells acquire the ability to break down their basal membraneproteolytically, to develop from a stationary polarised cell into anon-polarised cell capable of migrating in the tissue, to survive in thebloodstream and form metastases at remote sites (Liotta, et al., 1991;Liotta and Stetler-Stevenson, 1991).

Although deep changes in gene expression are involved in the manychanges in the architecture and behaviour of malignantly transformedcells, none of these newly acquired properties occurs only in invasivetumour cells. Attachment to the basal membrane, proteolysis thereof andmigration through the basal membrane and the underlying mesenchyme areimportant stages in normal processes, e.g. in the implantation of thetrophoblast, in movements of configuration during development of theembryo, in the development of the mammary gland and the reorganisationof epithelia during wound healing (Aznavoorian et al., 1993).

For a better understanding of the development and progression ofcarcinomas it is crucial to understand how the deregulation of thesenormal processes takes place in cell invasion and metastasisation.

Studies in recent years have contributed to an understanding of themolecular mechanisms involved in the modulation of the epithelialphenotype in normal and pathological situations (Reichmann, et al.,1992; Frisch, 1994). Moreover, exogenous polypeptide factors such asScatter Factor (SF)/Hepatocyte growth factor (HGF) andNew-Regulin/HER-Regulin play important roles in the changes in themigration and differentiation properties of epithelial cells (Birchmeieret al., 1993; Hartmann et al., 1994; Soriano et al., 1995). onlyrecently, Transforming Growth Factor 1 (TGFβ1) was identified as anotherpotent modulator of the phenotype of breast epithelial cells (Miettinenet al., 1994; Zambruno et al., 1995).

TGFβ1 belongs to a large super-family of multifunctional polypeptidefactors. The TGFβ family itself consists of three genes, TGFβ1, TGFβ2and TGFβ3, which have extremely high homology with one another. Inmammals the TGFβ-super-family also includes the various TGFβ genes aswell as the embryonic morphogenes, such as e.g. the family of theactivins, “Müllerian Inhibitory Substance”, and the bmp family (“BoneMorphogenetic Protein”), which play important roles both in regulatingembryo development and in the reorganisation of epithelia (Roberts andSporn, 1992). TGFβ1 inhibits the growth of many cell types, includingepithelial cells, but stimulates the proliferation of various types ofmesenchymal cells. In addition, TGFβs induce the synthesis ofextracellular matrix proteins, modulate the expression of matrixproteinases and proteinase inhibitors and change the expression ofintegrins. Moreover, TGFβs are expressed in large amounts in manytumours (Derynck et al., 1985; Keski-Oja et al., 1987). This strongoccurrence in neoplastic tissues could indicate that TGFβs are strategicgrowth/morphogenesis factors which influence the malignant propertiesassociated with the various stages of the metastatic cascade. TGFβsinhibit the growth of normal epithelial and relatively differentiatedcarcinoma cells, whereas undifferentiated tumour cells which lack manyepithelial properties are generally resistant to growth inhibition byTGFβs (Hoosein et al., 1989; Murthy et al., 1989). Furthermore TGFβ1 maypotentiate the invasive and metastatic potential of a breast adenomacell line (Welch et al., 1990), which indicates the role of TGFβ1 in thetumour progression. The molecular mechanisms underlying the effect ofTGFβs during the tumour cell invasion and metastasisation do, however,require further explanation.

The formation of breast cancer (mammary carcinoma) in humans involvesthe overexpression of (mutated or, more often, non-mutated) ras-genesand the overexpression of receptor-tyrosinekinases, which activate theRas-signal transmission pathway (De Bortoli et al., 1985; Kern et al.,1990; LeJeune et al., 1993).

SUMMARY OF THE INVENTION

The aim of the present invention was to provide new pharmaceuticalcompositions for tumour therapy.

The solution to the problem started from the following findings obtainedfrom the tests carried out:

1. The activity of TGFβ on the tumour cell, in cooperation with (i) theexpression of oncogenic Ras, with (ii) the overexpression of normal Rasor of receptor tyrosinekinases which activate the Ras signaltransmission pathway or with (iii) other oncogenes activated in thetumour cell, lead to a conversion of epithelial cells into fibroblastoidcells with invasive potential.

2. The autocrine production of TGFβ by the converted cells leads to themaintenance of the degenerate, invasive cell status.

3. Interruption of the transmission mediated by the TGFβ-receptor signalprevents “epithelial-fibroblastoid conversion” (EFC) and the concomitantinvasiveness and may change cells which have already undergone an EFCand are growing in a stably invasive manner back into epitheloid cellswhich are no longer growing invasively (fibroblastoid-epithelialconversion; FEC).

Within the scope of the present invention, the role of TGFβ1 in thenormal development of the mammary glands was investigated with a view toassessing possible side effects of TGFβ1 inhibitors.

Within the scope of the present invention, it was shown, on the onehand, that Ha-Ras-transformed breast epithelial cells (EpRas-cells)undergo a transition (conversion) from the epithelial to thefibroblastoid (or mesenchymal) state in the formation of tumours inmice. This transition is hereinafter referred to as EF-transition orEF-conversion (“Epithelial-Fibroblastoid Cell Conversion”, EFC). Such anEF-conversion has also been demonstrated in vitro. For this, EpRas cellswere cultivated in type I collagen gels. In the absence of serum thesecells developed into three-dimensional, cystic hollow structures, thewalls of which consisted of a single-thickness layer (monolayer) ofpolarised epithelial cells. TGFβ1 caused these same Ras-transformedcells to develop into disorganised strands consisting of spindle-shapedcells with fibroblastoid properties. In non-transformed epithelian cellsTGFβ1 was unable to cause such changes. The converted cells were highlyinvasive both in collagen gels and in chicken heart invasion assays.Surprisingly it was found that, once the fibroblastoid cells hadundergone the conversion, they themselves produced large amounts ofTGFβ1. If this self-produced TGFβ1 was inactivated by a TGFβ1neutralising antibody, the cells changed back into a polarised,epithelial phenotype. This cell behaviour indicates that the convertedfibroblastoid phenotype is maintained by TGFβ1, the TGFβ1 acting throughan autocrine loop.

It was also shown, within the scope of the present invention, that themechanism observed in vitro also applies in vivo: tumour cells which hadundergone an EF-conversion themselves produced TGFβ1. Moreover, TGFβ1 iscapable of triggering and sustaining the invasive phenotype ofHa-Ras-transformed breast epithelial cells in experimentally inducedtumours.

Moreover, it was shown within the scope of the present invention that inhuman tumours of various origins (kidney cell carcinoma, breast cancer)there were indications of the occurrence of “Epithelial-FibroblastoidCell Conversion” (EFC) (75% of the kidney cell carcinomas investigatedand 25-60% of the breast tumours coexpressed the general epithelialmarker cytokeratin and the mesenchymal marker vimentin). It was alsoshown that all these tumours themselves produce TGFβ1. This is anindication that the results obtained with the model system used in thepresent invention also apply to human tumours.

Fourthly, it has been shown within the scope of the present inventionthat total inhibition of the signal transmission induced by the TGFβreceptor can be achieved using a dominant-negative TGFβ-receptor chainII (TβRII-dn). Such expression of TβRII-dn led to the elimination of themalignant, invasive phenotype, not only in Ras-transformed mouse-breastepithelial cells, but also in a number of already mesenchymal,invasively growing carcinoma cell lines in humans and mice and to thecomplete inhibition of the formation of tumours or metastases obtainedby these lines in the experimental animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is thus based on the following findings:

Numerous mutations in protooncogenes and tumour suppressor genesparticipate in carcinogenesis (Vogelstein and Kinzler, 1993). However,little is known about how specific oncogenic mutations are connectedwith defined changes in the phenotype of the cell and the manner inwhich these changes then contribute to tumour cell invasion andmetastasisation. Within the scope of the present invention it was firstdemonstrated, using a model system, that the Ras-oncoproteindramatically changes the cell reaction of breast epithelial cells toTGFβ1 both in collagen gels and in developing tumours. This modifiedreactivity of the cells causes TGFβ1 to induce an EFC. Once converted,these fibroblastoid cells themselves produced high concentrations ofTGFβ1 and thus retained their own mesenchymal and invasive properties.

The theoretical validity of this principle could then be demonstrated ina number of unrelated tumour models in humans and mice. In these tumourcells, other oncogenes very probably take on the function of Ha-Ras. Ithas been shown that in all these cells both TGFβ1 and the interruptionof any existing autocrine stimulation by TGFβ1 dramatically influencesthe tumour cell phenotype: TGFβ also leads to an increase in invasivegrowth in these cells, whereas switching off the TGFβ-receptor or thesignal transmission pathways activated by it led to reformation of theEFC, i.e. to a fibroblastoid-epithelial conversion (FEC) and/or to lossof the invasive, tumour-producing cell phenotype.

The experiments carried out within the scope of the present inventionoriginally started from the observation that Ras-transformed mousebreast epithelial cells convert into invasive spindle cells duringtumour formation. Similar spindle cell tumours have been described inthe brain, skin, colon and breast, both in humans and in animal models(Buchmann et al., 1991; Guldberg, 1923; Sandford et al., 1961;Sonnenberg et al. 1986; Stoler et al. 1993). The origin of these spindlecell carcinomas is still unclear, although some researchers believe thatthese often highly invasive tumours constitute a separate class oftumour of fibroblastoid origin, whilst other authors assume that thesetumours are of epithelial origin.

In the model system used within the scope of the present invention thespindle cell tumours first used clearly originated from the epithelialdonor-cells injected into the animal. Spindle cells originating from thetumour survived the selection in G418 and expressed cell- andtissue-specific cytokeratins, confirming their donor cell status andtheir epithelial origin. Moreover, the tests carried out showed that theinjected epithelial cells and the converted fibroblastoid tumour cellscame from the same cell clone and re-integration of the retroviralvector into other sites of the genome could be ruled out as a possiblecause of the changes. What was almost more important was that thefibroblastoid phenotype of the converted cells was absolutely stableunder standard culture conditions and that the cells changed back topolarised epithelial cells efficiently after neutralisation of the TGFβ1activity. This rules out genetic or epigenetic changes being responsiblefor the cell conversion. The most probable explanation for the dramaticchange in the phenotype in vivo is that an interaction between theRas-transformed cells and mesenchymal cells surrounding them leads tothe conversion of the epithelial cells into fibroblastoid cells. Withinthe scope of the present invention it has thus been shown that EFC is amechanism which is relevant to carcinogenesis in certain tumours.

Within the scope of the present invention it has also been shown thatTGFβ1 induces EFC both in collagen gels and during tumour development.In the cell model used first, this TGFβ1-induced conversion remarkablyrequires the cooperation of an activated Ras protein; neither primarybreast epithelial cells nor the parental EpH4 cells underwentTGFβ1-induced EFC. From this it can be concluded that EFC is triggeredby a synergy of various signal transmission pathways which are activatedon the one hand by TGFβ1 and on the other hand by Ha-Ras. Thisassumption is supported by other findings which indicate that activatedRas proteins have similar effects on cells to members of the TGFβfamily. This applies e.g. to myogenic differentiation (Payne et al.,1987) and to the formation of the mesoderm (Whitman and Melton, 1992).In the heart muscle, TGFβ highly regulates genes associated with thegrowth of the embryonic heart regulated by haemodynamic loading. Theseeffects are at least partly mimicked by activated Ras (Parker et al.,1990; Thorburn et al., 1993), leading one to suppose that Ras and TGFβcan act synergistically, at least in some biological systems.

In connection with this it is interesting that the overexpression ofnormal and mutated Ras was observed in a considerable number of humancarcinomas, including breast cancers (De Bortoli et al., 1985; Hand etal., 1984; Slamon et al., 1984). Furthermore, autocrine production ofligands as well as overexpression and/or constitutional activation ofreceptor-tyrosinekinases which occur at the start of signal transmissionpathways including c-Ras (e.g. HER-1, HER-2), are frequent changes inbreast cancers (Kern et al., 1990; LeJeune et al., 1993). Since TGFβ1 isalso abundantly present in many human tumours (Derynck et al., 1985;Keski-Oja et al., 1987; Thompson et al., 1991) it may be concluded, onthe basis of the results obtained within the scope of the presentinvention, that Ras- and TGFβ1-induced signals also act synergisticallyin human tumours. As demonstrated by the results of the experimentscarried out within the scope of the present invention, the TGFβ-receptormay also control EFC and invasiveness in tumour cells transformed byoncogenes other than Ras. Another major finding within the scope of thepresent invention is thus that TGFβ cooperates with variousoncoproteins, including Ras, and tyrosinekinases, in regulating theplasticity of the polarised epithelial phenotype.

After serum-free (and TGFβ-free) cell culture in reconstituted collagengels, EpRas-cells exhibited a great capacity for organogenesis and ahigh degree of epithelial polarisation. The Ep-Ras cells, however,predominantly formed widened tubuli as well as alveolar cavities, incontrast to the narrow branching tubuli formed by the parentalEpH4-cells or by primary breast epithelial cells. This shows that theRas-oncoprotein on its own, in the absence of TGFβ, is capable ofmodifying the morphogenetic behaviour of epithelial cells to someextent. In other systems activated Ras has been described as having morepowerful effects on epithelial polarity (Eaton and Simons, 1995). Here,transformation with Ras led to disruption of the polar expression ofapical proteins, whilst the expression of basolateral marker proteinsremained unaffected (Schoenenberger et al., 1994). Since the experimentsdescribed above were carried out in the presence of FCS (which itselfcontains TGFβ1), however, it is difficult to compare them with theresults obtained within the scope of the present invention in which nosuch obvious polarity defects could be observed. Since exogenous TGFβ1completely destroyed the cell polarity, it is possible that the partialdestruction of the polarity in the abovementioned Ras-transformed cellsystems can be put down to the TGFβ1-concentrations present in theserum. Nonetheless the morphogenetic behaviour in EpRas-cells is changedslightly, possibly as the result of increased protease activity.

TGFβ completely destroyed the polarity of epithelial cells and causedthe cells to become spindle-shaped and invasive. These changes weredependent on the constant presence of TGFβ1, since the spindle cellsquickly changed back into polarised epithelial cells when TGFβ1neutralising antibodies were added. The most important conclusion fromthese results is that Ras-transformed cells can be switched back andforth by TGFβ1 between a quasi-normal and a highly tumorigenicphenotype. This intense phenotypical plasticity might be characteristicof invasively growing cells in general and might explain why invasivetumour cells often exhibit the migratory properties of fibroblasts.After extravasation these fibroblastoid, migratory cells should be ableto develop back into well differentiated secondary tumours in the newenvironment provided by remotely located tissue (see below). Increasedphenotypical plasticity is thus a characteristic of invasive tumourcells.

Another essential finding reached within the scope of the presentinvention is that EpRas cells have to undergo EFC in order to producesignificant amounts of TGFβ1 both in vitro and in vivo. It has beenshown that tumour cells are able to maintain their fibroblastoidphenotype by means of the autocrine production of TGFβ1 and that theautocrine TGFβ1 production and effect on the producing cell (autocrineloop) has to be interrupted in order to make the phenotypicalre-conversion of the cells possible. The ability of TGFβ1 to induce EFCand then efficiently maintain the invasive phenotype may also explainwhy the initially epithelial Ras-transformed cells changed progressivelyand uniformly into spindle cells during the tumour growth.

Shortly after their injection into mice, polarised Ras-transformedepithelial cells neither expressed nor released significant quantitiesof TGFβ1. As was established by hybridisation in situ andimmunohistochemistry, however, the stroma cells surrounding themicrotumour expressed the cytokine. These stroma cells could beidentified as fibrocyte and endothelial cells, but it must be presumedthat other cell types, such as macrophages and lymphocytes, wereprobably also present; all these cell types are known to produce andrelease TGFβ1. The most probable conclusion is that the effects of TGFβ1are regulated primarily at the level of their proteolytic activation.The primary regulation of TGFβ is carried out by factors which controlthe processing of the latent into the biologically active molecule.However, virtually nothing is known about the TGFβ activation in vivo.The protease plasmin can activate two cell types of latent TGFβ1 inco-culture systems, but only if two different cell types are in directcontact or close together (Antonelli-Orlidge et al., 1989; Sato et al.,1990). This close contact of different cell types should take place inthe system used within the scope of the present invention afterencapsulation of the tumours by the stroma and to an even greater extentif donor-tumour cells are mixed with stroma cells of the receiver animalduring the tumour development (FIG. 2B). Furthermore, thrombospondin(TSP), an extracellular matrix protein, activates latent TGFβ. In thiscase the activation takes place in the soluble phase and requires noproteolytic activity (Schultz-Cherry et al., 1994). In fact, the role ofthrombospondin in supporting the development of cancer and the increasedthrombospondin concentrations in malignant breast cancers have beenbriefly reported (Castle et al. 1991; Wong et al., 1992). It has thusbeen shown within the scope of the present invention that the autocrineproduction of TGFβ1, in cooperation with the oncoprotein Ha-Ras,maintains the fibroblastoid phenotype.

The findings and conclusions reached within the scope of the presentinvention led to the hypothetical model shown in FIG. 9. It ispostulated that the TGFβ1 which is relevant to the tumour formation isproduced primarily by infiltrating cells of the tumour stroma, such asfibrocytes, endothelial cells, lymphocytes and macrophages. Theinteraction of the tumour cells with the different cell types of thetumour stroma should trigger the efficient production and/or activationof TGFβ1. This should in turn cause the epithelial tumour cells tochange into the fibroblastoid and invasive phenotype. Thesefibroblastoid cells themselves then start to produce TGFβ1 which acts onthem in an autocrine loop and thus both maintains the fibroblastoidphenotype and also makes it easier to recruit other epithelial cells tothe EFC. Further mutations or selective mechanisms should cause some ofthese invasively growing cells to migrate into blood vessels and outagain and finally to form secondary tumours at remote sites. This modelconforms to findings which show that increased TGFβ1 expression is alsoinvolved in the progression to malignancy in a murine prostate cancermodel (Thompson et al., 1992; 1993).

The findings described hitherto were obtained in a combined in vitro/invivo model system using Ras-transformed mouse-breast epithelial cells.Within the scope of other tests, crucial aspects of this model (EFC,TGFβ1 production in the tumour) were detected in a large number ofprimary human carcinomas of the kidneys and breast. Thus, in themajority of all the kidney cell carcinomas investigated as well as in apercentage of the breast tumours investigated dependent on the degree ofmalignity, the occurrence of an EFC is demonstrated by the coexpressionof cytokeratin (general epithelial marker) and vimentin (mesenchymalmarker). Moreover, in all the tumours investigated, the production ofTGFβ1 by the tumour cells themselves has been demonstrated both at theprotein level by histochemical staining with anti-TGFβ-antibodies andalso at the mRNA level by in situ hybridisation and RT-PCR.

By means of another series of tests carried out within the scope of thepresent invention it has been shown that the TGFβ-receptor generallyassumes a central position in the regulation of EMT and invasive tumourcell growth. Not only in Ha-Ras transformed breast epithelial cells, butin a number of other tumours which originate from other epithelial typesand wherein it is not known which oncogenes take over the function ofHa-Ras, the TGFβ-receptor has been identified as the crucial regulatorof epithelial plasticity as well as of the invasive growth of the tumourcells. Thus, it has been possible to completely inhibit the invasivegrowth of two human carcinoma cell lines (kidney carcinoma line MZ 1795,nasopharyngeal carcinoma line KB) (presumably caused by secreted TGFβ1)in collagen gels by means of a neutralising anti-TGFβ1-antibody.

The proof of the above hypothesis was finally provided by means of adominant-negative TGFβ receptor (TβRII-dn). This TβRII-dn constitutes aso-called “kinase-dead” mutant of the receptor chain II which binds toendogenous receptors of type I, but cannot phosphorylate them. In thisway all the TβRII-dn-bound TGFβ-receptor chains of type I areinactivated because they cannot activate any signal transmission evenafter the binding of the ligand (TGFβ1) since the phosphorylation byreceptor chain II required for this is absent. If a dominant-negativeTGFβ-receptor of this kind is overexpressed in tumour cells, the entiresignal transmission proceeding from the TGFβ-receptor can be inhibitedin these cells. The expression of TβRII is thus suitable for simulatingthe activity of inhibiting TGFβ or inhibiting the signal transmissionpathway triggered by the activation of the TGFβ-receptor.

At first TβRII-dn was overexpressed in Ha-Ras-transformed mouse-breastepithelial cells (EpRas). All the clones obtained exhibited greatlydelayed tumour growth in nude mice. Moreover, the cells isolated fromsuch tumours had an epithelial phenotype and expressed epithelialmarkers (E-Cadherin, ZO-1) but no mesenchymal markers (vimentin). Thisshows that the expression of a TβRII-dn inhibited EFC during tumourformation.

After obtaining these results it was useful to check whether switchingoff the signal transmission of the TGFβ-receptor also works in tumourcells which have already undergone EFC and thus have a stablemesenchymal and invasive phenotype. The colon carcinoma line CT26 in themouse was selected as an example of such a cell line. This tumour cellline has a very marked tendency to form lung metastases rapidly aftersubcutaneous injection in mice, so that the animals die from the lungmetastases even after the primary tumour has been surgically removed ingood time. This cell exhibits mesenchymal morphology, grows intodisordered chains and strings of spindle-shaped cells in the collagengel and expresses no epithelial markers apart from basal cytokeratins.Instead the cells have a high vimentin expression. If thedominant-negative TGFβ-receptor (TβRII-dn) is overexpressed in thesecells, the cells form smaller or larger compact clumps in the collagengel and grow on plastic as epitheloid cells which form hemicysts (domes)and express large amounts of E-cadherin and ZO-1. The cells were thusobviously changed back, by the TβRII-dn, into cells with an epithelialphenotype (fibroblastoid-epithelial conversion, FEC).

A corresponding activity of the dominant-negative TGFβ-receptor(TβRII-dn) was also observed in vivo. When different, TβRII-dnexpressing clones of CT26 cells were injected into mice, the tumourformation was delayed by different amounts depending on the clone. Inmany clones tumour formation only occurred after 6-8 weeks, as opposedto 1-2 weeks in the case of animals injected with control CT26 cellswithout TβRII-dn. However, the activity of the TβRII-dn was even moredramatic when the primary tumours were removed from the mice at acertain size and the formation of metastases was expected. In thisexperiment metastases did not develop in any of the mice injected withTβRII-dn expressing CT26 cells (even after more than 18 weeks) whereasthe control animals died of lung metastases within 2-4 weeks afterexcision of the tumour.

The decisive conclusion from these experiments for the present inventionis that inhibiting the signal transmission mediated by the TGFβ-receptorcan not only prevent the occurrence of an EFC and the resultingacquisition of invasive properties, but also change any existing,invasively growing tumour cells back into a benign state in which theyare no longer invasive.

To sum up, the findings obtained within the scope of the presentinvention indicate that the increased sensitivity and altered reactivityof the cells, compared with the ability of TGFβ to modify the epithelialphenotype, generally represents a characteristic of epithelial tumourcells. This altered reactivity can be brought about byRas-(onco)proteins, but also by tyrosinekinases which activate Ras, aswell as by other as yet unknown oncoproteins. This alteredoncogene-induced mode of reaction to normal environmental signals, suchas e.g. those induced by TGFβ1, should lead to altered gene expressionin the tumour cell and also incorrect transmission or interpretation ofsignals between tumour and stroma cells. This abnormal “crosstalk”between tumour cells and their immediate environment would appear to bethe driving force for what is commonly known as tumour progression.

Furthermore, the results of the experiments show that activated Ras,overexpressing receptor-tyrosinekinases which activate the Ras signaltransmission pathway as well as other, possibly unknown oncogenesco-operate both in the normal development and also in the carcinogenesiswith the TGFβ1-receptor. This would appear to involve processes such asthe induction/activation of stromal TGFβ1 by the interaction ofepithelial and mesenchymal cells as well as EF conversion, induced andmaintained by TGFβ1.

The main difference between normal and oncogene-transformed tumour cellsshould therefore be as follows: TGFβ1 has a physiological, strictlyregulated function during the morphogenesis of normal cells. In thetumour cell the transformation by oncogenes causes degeneration of thefunction of TGFβ1, i.e. constitutive, highly abnormal morphogeneticchanges are triggered in the cells.

The present invention thus relates to a pharmaceutical compositioncontaining as active compound a substance which inhibits the activity ofTGFβ on tumour cells of epithelial origin, for treating epithelial,invasive tumour diseases which are characterised by a reversibletransition of the cells from an epithelial, non-invasive state into aninvasive state.

In one embodiment of the invention the pharmaceutical composition alsocontains substances which inhibit the expression of oncogenic Ras and/orthe overexpression of normal Ras or the activity of Ras-activatingreceptor tyrosinekinases in the cells.

In epithelial invasive tumour diseases the tumour cells have anincreased phenotypical plasticity, i.e. they are able to undergotransitions from the epithelial, non-invasive state to thefibroblastoid, invasive state (EF conversion) and vice versa (FEconversion).

The substance which inhibits the activity of TGFβ on the cells or thesignal transduction mediated by activation of the TGFβ-receptor ishereinafter referred to as “TGFβ inhibitor”.

TGFβs, like the other members of the TGFβ super-family ofmultifunctional polypeptide factors such as e.g. activins, BoneMorphogenetic Proteins (bmp's), etc., exert their effect by binding tospecific cell surface receptors. The type I and type II TGFβ receptorsform heterodimeric complexes after binding of the ligand, therebyinitiating the signal transmission. The type II receptors, which areassigned to the group of the receptor serine/threonine-kinases in termsof their activity, bind the ligands, but require association with thetype I receptors which constitute serine-threonine-kinases in order tobe able to pass on the signal obtained from the ligand. Whereas the typeII receptors are responsible for the ligand specificity, thefunctionally different type I receptors heterodimerise with several typeII receptors. In this ligand-induced heterodimerisation the typeII-receptor chains phosphorylate the type I receptors onserine/threonine groups and thereby activate them. This cooperation ofthe type II-receptor with a particular type I-receptor causes activationof specific signal transmission pathways and as a result leads to atranscriptional response to the signals transmitted to the cell by theligands.

The activity of a TGFβ inhibitor is based on the fact that it blocks thecell response triggered by the receptor activation, i.e. it prevents theTGFβ-receptor system from being activated and hence the cell signaltransmission pathway from being actuated.

Since it is the type I receptors which are responsible for the specifictranscriptional response which eventually produces the fibroblastoidphenotype after the binding of the ligands to the type II receptor, andto the type I receptor, the type I receptor represents one of the targetmolecules for the TGFβ inhibitor. Because of the need forphosphorylation of the type I receptor by the type II receptor (and onthe basis of the results obtained with dominant negative type IIreceptors whose serinekinase activity has been destroyed by mutation,the type II receptor is also a possible target molecule for inhibitors.

Other mechanisms for the activity of a TGFβ inhibitor are thus based onpreventing the interaction between the ligand TGFβ and the type IIreceptor, preventing the signal transmitted from the type II receptor tothe type I receptor which brings about the activation of the type Ireceptor. Finally, blocking the binding of TGFβ to the type I receptor,inhibiting the activity of the type I receptor or inhibiting an effectormolecule of the signal transmission pathway activated by the type Ireceptor are all possible methods of attacking inhibitors.

Examples of inhibitors are antibodies which neutralise the TGFβ,particularly monoclonal antibodies, TGFβ antisense-RNA molecules(Fakhrai et al., 1996) or dominant-negative TGFβ receptors of type I orII.

The invention relates, according to a further aspect, to screeningprocesses for identifying pharmacologically active substances fortreating epithelial, invasive tumour diseases which are characterised bya reversible transition of the cells from an epithelial, non-invasivestate into an invasive state.

One method of finding suitable, particularly low-molecular, inhibitors,comprises determining, in a first step, which of the type I receptors isresponsible for the transition from the epithelial to the fibroblastoidstate of the cells. To do this, the EpRas-cell line used within thescope of the present invention (or one of the other cell lines usedwhich are capable of bringing about the EF conversion or have alreadyundergone one), is interrogated to see which TGFβ-type I/II receptor itexpresses. This interrogation may be carried out by RT-PCR (“ReverseTranscriptase Polymerase Chain Reaction”) using oligonucleotides,derived from known TGFβ-type I or type II receptors, as PCR primers inorder to amplify the relevant receptor-DNA from EpRas-DNA and thusidentify the TGFβ-type I or type II receptor expressed in these cells.The experiments described in the Examples with a dominant-negativemutant of the human type II receptor TβR-II (only this chain occurs inall the known receptors for TGFβ1,2,3; Wrana et al., 1992, Wrana et al.,1994) confirm that this TGFβ-type II receptor subtype (as such) isnecessary, directly or indirectly, for the signal transmission whichleads to the EF conversion. This TGFβ-type II receptor subtype thusconstitutes one of the target molecules for the TGFβ inhibitor. Thissatisfies an esential precondition for establishing a cellular orbiochemical screening assay which can be used to screen specifically forsubstances which inhibit this target molecule.

Next, an investigation is carried out in a cell which is undergoing theEF conversion or in which the EF conversion can be reversed byinhibiting the TGFβ receptor signal transmission to determine which ofthe processes taking place in the cell by EF conversion or reversalthereof is most suitable for establishing a screening assay.Appropriately the EpRas cells used in the Examples or the CT26 cellswhich are also well characterised within the scope of the presentinvention may be used.

The effects to be expected can be divided into two groups: the firstgroup includes the effects of TGFβ on normal mesenchymal and epithelialcells, e.g. in wound healing, described in the literature. The secondgroup of changes are those which occur in particular with the activityof TGFβ on transformed cells (such as e.g. in the experiments describedwithin the scope of the present invention). Whereas the TGFβ effects ofthe first group can be adduced for a primary HTS screen (“HighThroughput Screen”), any inhibitor candidates found must be testedwithout fail for their inhibiting activity on the TGFβ effects of thesecond group.

The TGFβ effects of the first type include i) the induction ofextracellular matrix proteins, such as fibronectin, laminin, elastin;ii) the induction of the protease inhibitor PAI (Plasminogen ActivatorInhibitor) and hence the inhibition of cell protease activity, and iii)an inhibition of cell growth and induction of programmed cell death(apoptosis) in certain cell types. These include in particular normalepithelial cells as well as only slightly degenerate, essentially stillepitheloid tumour cell lines. The induction of PAI-1 expression as wellas the TGFβ-induced apoptosis are possible procedures which may be usedto design a cell assay system for screening substances which may act asTGFβ receptor inhibitors. The effect chosen is used directly as a systemfor demonstrating the inhibiting activity of the substance.

In order to find out whether the EF conversion in the EpRas-cell lineused within the scope of the present invention triggered by theactivation of the TGFβ receptor system is transmitted via the same typeI/type II receptors as the induction of PAI (or another moleculeregulated by TGFβ) in untransformed cells, e.g. in the normal startingcell line EpH4 (also used within the scope of this invention), it ispossible to check e.g. whether the induction of PAI (or anothermolecule) or the growth inhibition which is very marked in this cellline is blocked by a dominant-negative mutant of the same type I or IIreceptor which also blocks the EF conversion. In the case of the CT26cells which overexpress the dominant-negative type II receptor(TβRII-dn), it has been shown that, in the TβRII-dn expressing CT26clones which reverted to epithelial cells, activation of a PAI-1promoter-controlled reporter gene was completely inhibited by TGFβ-1.The extent of the PAI-I inhibition of the inhibition of reporter geneexpression by PAI-I correlated directly with the ability of thedifferent clones to form tumours in the animal. Moreover, a special CT26clone, which had recovered complete TGFβ-1-inducibility of the PAI-1promoter-reporter gene construct after lengthy passage in vitro(presumably by repressing the TβRII-dn expression) also regained theability to form metastasising tumours in the mouse.

The confirmation of the correlation between EFC, tumour formation andTGFβ receptor type II function using the TβRII-dn experiments providesthe prerequisite for a screening assay based on a PAI-1 reporter genetest cell. This test cell, which is a human or animal cell, is stablytransformed with a plasmid, in which a reporter gene, e.g. theluciferase gene, is under the control of the regulatory sequence of thePAI gene (or a gene which codes for another molecule regulated by TGFβ,e.g. for an extracellular matrix protein). The test cell is alsotransformed with the human type I or type II receptor, which was shown,after further tests, to be most efficient both at triggering the EFconversion and also at inducing PAI or another molecule regulated byTGFβ. The human TGFβ type II receptor used for the construction of theTβRII-dn is one of the possible target molecules for a TGFβ inhibitor.The control cell used is expediently a parallel-cell clone in which thePAI-1-promoter controlled reporter gene is activated by another receptornot related to the TGFβ receptor (e.g. members of the FGF (fibroblastgrowth factor) receptor-tyrosinekinase family).

If a substance which wholly or partially inhibits the TGFβ-inducedreporter gene expression is found in a screening assay of this kind, itcan be concluded that either the selected ligand-activated type I/typeII receptor or the signal transmission mediated by this receptor isblocked by this substance. The same substance should not have anyinfluence on the slight basal reporter gene expression in the controlcell in which the reporter gene has been activated not by TGFβ, but byFGF. The test systems in which the reporter gene activation is measuredcan be used in robotised High Throughput Screen (HTS) processes.

A second possible way of measuring the blocking of the TGFβ receptorfunction by test substances can easily be measured by the removal of thegrowth inhibition and apoptosis brought about by TGFβ. Since TGFβefficiently induces apoptosis in normal EpH4 cells under certainconditions, effective inhibitors of the TGFβ receptor should act assurvival or growth stimulating factors. EpH4 cells in which anotherapoptosis-inducing receptor has been expressed may be used as controlcells. The Fas receptor, which efficiently induces apoptosis invirtually all cell types after the binding of a special Fas ligand, isparticularly suitable. The removal of an apoptotic effect by effectiveTGFβ receptor inhibitors has the advantage that it can easily bemeasured in commercially obtainable test systems (e.g. in the MTS assaywhich detects the number of live, metabolically active cells), and thattoxic substances (which cause rather than prevent cell death) can easilybe identified as such. Thus, this test system is also suitable for HTSprimary screens.

Another possible cell assay system with which substances can be testedfor their inhibiting activity on the EF conversion triggered byactivation of the TGFβ receptor system, is based on the expression ofproteins which are characteristic of the fibroblastoid cell type afterEF conversion and are thus an indicator of the occurrence of EFconversion. One example of this is vimentin (Reichmann et al, 1992): ithas been shown within the scope of the present invention that expressionthereof goes hand in hand with the EF conversion triggered bycooperation of Ras and TGFβ. Other examples of other markers of thefibroblastoid phenotype are the loss of the expression of E-CadherinmRNA as well as the de-novo expression of fibronectin and diverseproteases (UPA, TPA, Reichmann et al, 1992). A suitable test celltransformed by Ras or another oncogene is transformed with a plasmid inwhich a reporter gene is under the control of the vimentin gene promoteror of promoters of one of the other fibroblastoid marker genesmentioned. The modulation of the reporter gene expression by a testsubstance should then correlate with the modulation of the EC conversionbrought about by the same inhibitors.

Another possible way of finding substances which inhibit the activationof the TGFβ receptor system uses the expression of TGFβ itself as adetection system. This assay principle is based on the finding reachedwithin the scope of the present invention that the activation of theTGFβ receptor system in oncogene expressing cells by the ligand TGFβcauses the autocrine production of TGFβ which acts on the cells in anautocrine loop. In an assay of this kind, which can detect both theactivation of the TGFβ receptor system and also the induction of theautocrine TGFβ loop brought about by the expression of Ras (the activityof substances which inhibit TGFβ expression, in a test of this kind, dothis on the basis of their effect on the activation of the TGFβ receptorsystem and their effect on Ras), the cells contain a reporter geneconstruct which is under the control of the TGFβ gene promoter (Kim etal, 1989).

Biochemical assays in which TGFβ inhibitors are identified, the activityof which is based on the fact that they inhibit the TGFβ signaltransmission pathway, may be carried out as follows, for example: in anassay format the autophosphorylation of the TGFβ receptor type II or thecytoplasmic domain thereof which contains the kinase domain is measuredin vitro on serine or threonine groups, in the presence and in theabsence of test substances (potential TGFβ inhibitors), a kinase assayof this kind being carried out using methods known from the literature,e.g. as described by Lin et al., 1992, or Braunwalder et al., 1996, andusing a receptor (or a domain thereof) prepared by recombinant methods,e.g. in E. coli. In an alternative assay format, the ability of the TGFβreceptor type II to phosphorylate the TGFβ receptor type I or itsso-called GS domain (Wrana et al., 1994), is measured, again accordingto the known principle of kinase assays, in the presence and absence ofpotential inhibitor. The modification of an assay of this kind for aHigh Throughput Format can be carried out using commercially availabletechnologies such as filter plates, FLASH plates (Amersham) or SPA(Scintillation Proximity assay)-Beads (Amersham).

In one of the test systems described, inhibitors of the TGFβ receptorfound in the primary screen are expediently tested for their specificityin secondary screens. This can be done particularly by direct inhibitionof the TGFβ-dependent EF conversion of EpRas cells in collagen gels.Another possibility is the incubation of converted EpRas cells (e.g.from mouse tumours) plated out at low density on plastic dishes with theinhibitor of the TGFβ receptor found. Effective substances shouldtrigger the conversion of fibroblastoid into epithelial cells even inthe presence of TGFβ. The same substances should causere-epithelialisation (FE conversion) in CT26 cells. Finally,particularly suitable active substances in mice which were injected withCT26 cells can be tested to see whether they slow down the growth of theprimary tumour or metastasisation after excision of the primary tumour.

The substance which inhibits the expression or the function of oncogenicRas and/or the overexpression of normal Ras (or the consequences of thisoverexpression) and/or the activation of normal Ras by receptortyrosinekinases in the cells is hereinafter referred to as “Rasinhibitor”.

Ras inhibitors for the purposes of the present invention either inhibitRas directly, by inhibiting the activation/function of Ras itself or byinhibiting the activation/function of a Ras-effector molecule which actsbelow Ras in the Ras signal transmission pathway. Examples areinhibitors of Raf, such as Raf antisense-oligonucleotides (Monia et al.,1996). For cases where the activation of Ras cannot be put down to achange in Ras itself, but it due to the constitutive activation ofreceptor-tyrosinekinases acting above Ras, inhibition of Ras-activationcan also be brought about by inhibiting these receptors. Examples ofreceptors of this kind are the receptor-tyrosinekinases EGF receptor(“Epidermal Growth Factor Receptor”) and homologous receptors such asHER-2, HER-3 or HER-4. Examples of chemical compounds which inhibit theEGF receptor can be found in WO 96/07657. Known Ras inhibitors aremonoclonal antibodies (Furth et al., 1982), dominant-negative mutants(Stacey et al., 1991; Quilliam et al., 1994) and antisense-RNA. Examplesof low-molecular Ras inhibitors are inhibitors of Ras-Farnesyltransferases (Kohl et al., 1993; Kohl et al., 1994; Kohl et al., 1995).

In order to screen for other low-molecular Ras inhibitors, genes codingfor mutations of the Ras proteins H-Ras, K-Ras or N-Ras, which lead toconstitutive activation of Ras, are introduced into mammalian cells,e.g. by means of retroviral vectors, and the selective cytotoxicactivity of test substances on the ras-transformed cells is determined.A suitable method of identifying ras inhibitors is described e.g. in derEP-A 604 181.

Examples of Ras-transformed cell lines which may be used as test cellsfor the identification of Ras inhibitors, have also been described byAndrejauskas and Moroni, 1989, as well as by Jenkins et al., 1993.

Ras inhibitors can also be identified with an assay based on theEpRas-cell line used within the scope of the present invention. Forthis, the cells contain a reporter gene construct in which the reportergene is under the control of the regulatory sequence of the TGFβ gene.First of all TGFβ is applied to the cells in order to bring about the EFconversion. Then the cells are treated with the test substances. Testsubstances which are capable of inhibiting the activity of the reportergene can be assumed to be Ras inhibitors. This can then be confirmed insecondary screens in which the substances are investigated to seewhether they can inhibit the TGFβ-induced EF conversion of EpRas cellsin collagen gels or reverse the EFC which has already taken place.

The pharmaceutical compositions according to the invention can be used,firstly, to prevent the cells from changing into the fibroblastoid stateand becoming invasive, thus preventing or reducing their tumorigenicity.Secondly, the pharmaceutical compositions according to the invention canalso be used to bring about the conversion of existing fibroblastoid andinvasively growing tumour cells into non-malignant or less malignantepithelial cells.

The pharmaceutical composition according to the invention may be used onthe one hand to prevent the transformation of the cells from theepithelial, non-invasive state into a fibroblastoid, invasive state. Oneexample of this is its administration after surgical removal of aprimary tumour to prevent any tumour cells present from becominginvasive and producing further tumours by metastasisation. Moreover thepharmaceutical composition according to the invention can also slow downtumour growth by the same mechanism, as has been shown with the aid ofthe TβRII-dn expressing CT26 cells.

The pharmaceutical composition according to the invention may, on theother hand, be used to reverse an EF conversion of the cells which hasalready taken place. Once the conversion has taken place, TGFβ maintainsthe fibroblastoid state by means of an autocrine loop. Theadministration of a TGFβ inhibitor on its own in this case switches offthe autocrine loop and thus reverses the fibroblastoid, invasive stateof the cell into the normal, epithelial state. However, this reversal istemporary, and there is no fundamental change to the transformed stateof the cell brought about by Ras or other oncogenes. This means thatwhen the TGFβ inhibitor is removed the EF conversion could start upagain. If, on the other hand, an oncogene inhibitor, e.g. a Rasinhibitor or an HER-1/2 inhibitor, is administered, possibly in additionto the TGFβ inhibitor, the transformed state of the cell is cancelled,the cell behaves like a normal epithelial cell and reactscorrespondingly normally to TGFβ, i.e. the effect of TGFβ on the cellcannot bring about EF conversion and even leads to growth inhibition ofthe tumour cell.

The conjecture that TGFβ (receptor) inhibitors could cause slowing downor even inhibition of tumour growth is supported by the following stateof affairs: most tumours constantly produce TGFβ (see below) which isreleased into the environment and has an immunosuppressant effect there,i.e. inhibits the function of cytotoxic T-lymphocytes and other cells ofthe immune system. If the TGFβ receptor inhibitor causes thetransformation of invasive tumour cells into non-invasive, moreepithelial cells, these should switch off the secretion of TGFβ and thusbe more easily attacked and lysed by cytotoxic T cells.

In order to achieve optimal activity, the pharmaceutical compositionaccording to the invention preferably contains a combination of TGFβinhibitor and Ras inhibitor.

In the transition of epithelial cells into the fibroblastoid state,fibroblastoid marker proteins, e.g. vimentin, are expressed moreintensely. The increase in the expression of these markers (see below)is thus one of the diagnostic parameters for tumour diseases which canbe treated using the pharmaceutical composition according to theinvention.

These tumour diseases include adenocarcinomas of the breast (Heatley etal., 1993), kidney cell carcinomas (Beham et al., 1992), carcinosarcomasof the breast (Wargotz and Norris, 1989), carcinosarcomas of theoesophagus (Guarino et al., 1993) or of the female genital tract (deBrito et al., 1993), epitheloid sarcomas as well as spindle cellcarcinomas of various locations, e.g. lung carcinomas with spindle cellcomponents (Matsui et al., 1992) or spindle cell carcinomas of the gallbladder (Nishihara et al., 1993).

The pharmaceutical composition according to the invention is preferablyused to treat breast tumours and kidney cell carcinomas.

The pharmaceutical compositions according to the invention areadministered to humans in doses of 0.01 to 100 mg/kg body weight,preferably 0.1 to 15 mg. Apart from the active compounds thepharmaceutical composition contains the usual inert carriers andexcipients. The skilled person will find methods of formulatingpharmaceutical preparations in the relevant textbooks, such asRemington's Pharmaceutical Sciences, 1980.

FIGURE SUMMARY

FIGS. 1A-1D conversion of EpRas cells into fibroblastoid cells duringtumour formation in mice.

FIG. 1A is a schematic diagram illustrating the strategy which was usedto study the epithelial-fibroblast conversion (EFC) of Ras cells invivo.

FIG. 1B is a photomicrograph showing cells of the clone Ep5 beforesubcutaneous injection.

FIG. 1C is a photomicrograph showing that Ep5 cells isolated from atumour 28 days after injection.

FIG. 1D is a Southern Blot analysis of the EpRas-clone (Ep5): (i) beforeinjection (Ep5 plastic), (ii) removed from the tumour (Ep5, tumour), and(iii) removed from the tumour and recultivated for 5 days in G418 (Ep5,ex tumour).

FIGS. 2A-2H Conversion epithelial/mesenchymal (EFC) during tumourdevelopment: time scale and behaviour of donor and receiver cells: after3 days (FIGS. 2A and 2E), 7 days (FIGS. 2B and 2F), 15 days (FIGS. 2Cand 2G) and 28 days (FIG. 2H). FIG. 2D is a photomicrograph showingparental EpH4 cells 15 days after subcutaneous injection.

FIGS. 3A-3D Organogenesis and epithelial polarity are destroyed by serumor TGFβ1.

FIG. 3A is a photomicrograph showing Ep4H cells in a serum-free collagengel.

FIG. 3B is a photomicrograph showing EpRas cells in a serum-freecollagen gel.

FIG. 3C is a photomicrograph showing cells after the addition of 10%FCS.

FIG. 3D is a photomicrograph showing EpRas cells grown with TGFβ1 (5ng/ml).

FIGS. 4A-4F TGFβ1 destroys the cell polarity in Ras-transformed breastepithelial cells.

FIG. 4A is a transmission electron micrograph of EpRas cells inserum-free collagen gel.

FIG. 4B is a photomicrograph of a frozen section through an alveolarcyst formed by EpRas cells in serum-free collagen gel.

FIG. 4C is a photomicrograph of a Lowicryl section through an alveolarcyst formed by EpRas cells in serum-free collagen gel.

FIG. 4D is a transmission electron micrograph of disordered strings ofEpRas cells after treatment with TGFβ1.

FIG. 4E is a photomicrograph of a frozen section through disorderedstrings of EpRas cells after treatment with TGFβ1.

FIG. 4F is a photomicrograph of a Lowicryl section through disorderedstrings of EpRas cells after treatment with TGFβ1.

FIGS. 5A-5D Fibroblastoid EpRas cells are highly invasive in the chickenembryo heart invasion assay. FIGS. 5A-5D are photomicrographs of in vivofluorescence-labeled cells co-cultured for 7 days with chicken embryoheart fragments: non-tumorogenic epithelial starting cells (EpH4 cells)(FIGS. 5A-5B), non-converted epithelial EpRas cells (FIG. 5C), andconverted fibroblastoid cells after TGFβ1 treatment (FIG. 5D).

FIGS. 6A-6F TGFβ1 maintains the fibroblastoid phenotype of convertedEpRas cells via an autocrine loop. FIGS. 6A-6D are photomicrographs of acell clone, from fibroblastoid cells isolated from a tumour and grown inmedium containing 1% FCS, on day 1 (FIG. 6A), day 3 (FIG. 6B), day 5(FIG. 6C) and day 10 (FIG. 6D). FIGS. 6E-6F arc photomicrographs of thesame cells after a further 8 days in collegian gels, in the absence(FIG. 6E) and in the presence (FIG. 6F) of TGFβ1 neutralizingantibodies.

FIGS. 7A-7B Converted EpRas cells produce high concentrations of TGFβ1.

FIG. 7A shows a semi-quantitative PCR analysis for TGFβ1 -mRNA.

FIG. 7B show TGFβ1 concentrations in cell culture supernatants asmeasured by Western Blot and ELISA

FIGS. 8A-8F TGFβ1 triggers the transition from the epithelial to thefibroblastoid state as well as the invasiveness of the cells inexperimentally induced tumours.

FIGS. 8A-8B are photomicrographs of frozen sections of a tumour on day4.

FIGS. 8C-8D are photomicrographs of frozen sections of a tumor on day15.

FIG. 8E is a photomicrograph of EpRas cells injected subcutaneously intonude mice without 3-Elvax Slow Release Pellets charged with recombinant(active) TGFβ1.

FIG. 8F is a photomicrograph of EpRas cells injected subcutaneously intonude mice with 3-Elvax Slow Release Pellets charged with recombinant(active) TGFβ1.

FIG. 9 model for the activity of TGFβ1 in tumour development

FIGS. 10A-10D TGFβ1 induces in vitro morphogenesis and apoptosis innormal mammary gland epithelial cells.

FIG. 10A is a photomicrograph showing that, in the absence of additionalTGFβ1, the cells do not form any tubular structures.

FIG. 10B is a photomicrograph showing that, in the presence of 0.1 ng/mlof TGFβ1 the cells form branched structures, but these branchedstructures lack lumina.

FIG. 10C (lower magnification on the left, higher magnification on theright) is a photomicrograph showing that higher concentrations of TGFβ1cause cell death (apoptosis).

FIG. 10D is a photomicrograph showing that, if the TGFβ1 is removed, onday 7 by washing, from cultures having the structures shown in FIG. 10C,the cells form distinct hollow structures.

FIG. 11 In vivo expression of TGFβ1 during the formation of the normalbreast

FIG. 12 In vivo expression of TGFβ1 during the breakdown of the fullydeveloped mammary gland after ablactation

FIG. 13 inhibition of the invasivity of human tumour cells byTGFβ-neutralising antibodies

FIG. 14 expression of a dominant-negative TGFβ-receptor (TβRII-dn) inRas-transformed breast epithelial cells inhibits EFC and tumour growth

FIG. 15 expression of TβRII-dn in mouse-colon carcinoma cells (CT26)prevents growth in collagen gels and invasivity in vitro

FIG. 16 expression of TβRII-dn in CT26 cells inhibits metastasisation invivo

FIG. 17 TβRII-dn expressing CT26 cells are incapable of formingmetastases in the lung even after intravenous injection

FIG. 18 expression of a PAI-1-promoter-reporter construct in CT26− andCT26+TβRII-dn cells ]

EXAMPLES

In the following Examples, the following materials and methods were usedunless otherwise stated:

a) Cell Culture

EpRas cells were prepared by infecting the parental breast epithelialcell line EpH4 (a subclone of the spontaneously immortalised breastepithelial cell line Ep1 (Reichmann et. al, 1992) selected for thestrong impression of a polarised phenotype) with a helper-free, v-Ha-Rasexpressing retroviral vector (Redmond et al., 1988). The selection andexpansion of the polarised epithelial clones was carried out asdescribed by Reichmann et al., 1992. For this the cells were cultivatedon plastic dishes in growth medium (Dulbecco's modified Eagles medium;(DMEM), containing 10% FCS (Boehringer Mannheim) and 20 mM HEPES, andsubcultivated in a ratio of 1:3 twice a week. For the induction ofhemicyst(dome) formation EpRas cells and cells of the parental line EpH4were cultivated at high density for one week without subcultivation.

The human tumour cell lines MZ 1795 (kidney carcinoma; Seliger et al.1996) and KB (nasopharyngeal carcinoma ATCC CCL17; Derynck et al., 1985)were obtained from the ATTC. They were cultivated in the same medium asthe mouse EpH4 cells.

The mouse colon carcinoma line CT26, the establishing of which wasdescribed by Brattain et al., 1980, was also cultivated in the samemedium as the mouse-EpH4 cells.

In order to express the human, dominant negative TGFβ receptor type II(TβRII-dn, Wrana et al., 1992) in mouse-EpH4 and CT26 cells, thecorresponding cDNA (Wrana et al., 1992) was inserted in the helper-freeretrovirus vector pbabe-puro (Morgenstern and Land, 1990). Theretrovirus-DNA was transfected into BOSC 23 packaging cells (Pear et al,1993) and the virus-producing, Mitomycin-C treated BOSC 23 cellscocultivated with EpH4 or CT26 cells. Infected clones were selected withG418 and expanded in Dulbecco's modified Eagles medium (DMEM),containing 20% FCS (Boehringer Mannheim) and 20 mM HEPES. The expressionof the TβRII-dn protein was detected in the Western Blot using ahaemagglutinin (HA) epitope present in the construct.

b) Growth of Organotypical Cell Structures in Collagen Gels

Semiconfluent cultures of the cells to be analysed were trypsinised andadjusted to a final concentration of 4×10⁴ cells per ml with ice-coldgrowth medium. Equal volumes of the cell suspension and an acidifiedsolution of rat's tail collagen type 1 (Sigma) were mixed at 4° C.,applied to 35 mm tissue culture dishes and incubated for 30 min at 37°C., in order to allow the solution to set to a gel. To allow the cellsto form organotypical structures, the collagen gels were covered with aserum-free medium (MEGM; Promocel) containing bovine pituary extract(BPE), recombinant epidermal growth factor (EGF), hydrocortisone andinsulin (in the concentrations recommended by the manufacturer). Wherestated, 5 or 10% FCS or 5 ng/ml recombinant TGFβ1 (literature) wereadded. The medium covering the collagen gels was changed every two days.In order to neutralise the TGFβ1 produced by the cells or cellstructures in the collagen gels a monoclonal antibody against TGFβ1(Genzyme) or control antibody is used in concentrations of up to 50μg/ml.

c) Tumour Induction in Mice and Re-isolation of Cells from Tumours orCollagen Gels

Confluent EpRas or EpH4 cells were trypsinised and counted. Then 10⁵cells, suspended in 0.1 ml PBS, were injected subcutaneously or into themammary gland of 5 week old BALB/c mice or nude mice. The mice werekilled after different lengths of time (between 3 and 28 days) and thetumours (or tissue zones which the injected cells contained) wereexcised. For the subsequent histological analysis the tissue wasimmediately flash-frozen in liquid nitrogen. To isolate the tumour cellsfor further growth in the tissue culture, the tissue was cut into smallpieces under sterile conditions using two opposing scalpels and digestedwith 2 mg/ml collagenase type 1 (Sigma) for 1 hour at 37° C. In order toremove the remaining host cells which lacked the retroviral neomycin orhygromycin resistance markers of the donor cells, the cells obtainedfrom the tumours were grown for the first 5 days in the presence of G418or hygromycin. The collagen gels were digested with collagenase in asimilar manner, in order to isolate the cells for the subsequent tissueculture.

For tumour induction with CT26 cells or CT26−TβRII-dn cells, 1×10⁶ cellsper animal were injected subcutaneously into nude mice under the skin ofthe back. The size of the detectable tumours was determined every 3 daysand the animals were killed when the tumours exceeded a sze which wastolerable for the wellbeing of the animals.

In another experiment 1×10⁶ cells per animal were injected into syngenicmice (Balb/C). After the primary tumour reached a size of 4 cm3 thetumours were surgically removed so that there was no tumour tissueremaining at the site of the operation. The mice were then furthermonitored and after their death the presence of lung metastases wasinvestigated.

In a last experiment to demonstrate the ability of the CT26− andCT26−TβRII-dn cells to colonise the lung from the circulation, 5,000 and50,000 cells of both types were injected i.v. into the caudal vein ofsyngenic Balb/C mice. After their death the mice were then examined asbefore for the presence of lung metastases.

d) Antibodies

Rabbit antisera against cytokeratin have been described by Reichmann etal., 1992. Rabbit antiserum and monoclonal rat antibodies againstE-Cadherin were prepared as described by Kemler, 1993 and the relevantliterature cited therein (Kemler, 1993). Rabbit antiserum whichrecognises neomycin phosphotransferase was prepared by bacteriallyexpressing neomycin phosphotransferase, purifying it and injecting itinto rabbits. After a reasonable time rabbit serum was obtained and usedneat for immune staining. The monoclonal mouse antibody against vimentinV3B (Boehringer Mannheim), the monoclonal rat antibody against ZO-1(Chemicon), the monoclonal mouse antibody against TGFβ 1-3 (Genzyme),the TGFβ 2,3-antibody (Genzyme), the polyclonal antiserum againstactivated TGFβ (Promega), the TGFβ-neutralising polyclonal rabbitantibody (R&D) as well as the monoclonal TGFβ-antibody (Genzyme) wereobtained commercially.

e) Sections and Immunofluorescence

In order to obtain the optimum biological activity from RNA and proteinsin the excised tissues and collagen gels, the tumour material and thecell structures containing collagen type I gels were flash-frozenimmediately after isolation in liquid nitrogen. Before freezing thecollagen gels were soaked for 2 min in medium containing 5% DMSO inorder to prevent cell damage as a result of the formation of icecrystals. Cells grown on plastic or frozen sections prepared fromtumours or collagen gels were fixed and made pervious for 15 min at −20°C. using acetone/methanol, mixed in the ratio 1/1, air-dried and storedat 4° C. The incubation with the first antibody was carried out for 1 hat 37° C. in PBS which normally contained gelatin, BSA and Tween 20(0.2% each), in order to prevent non-specific antibody staining. Thecells or sections were then covered in Moviol 1-88 (Hoechst) andexamined with a Zeiss Axiophot fluorescence microscopy. The photographswere prepared either conventionally or by computer-aided methods using aKaf 1400 CCD camera (Photometric) and the Adobe Photoshop 3.0 picturedeveloping program.

In order to detect cytokeratin, vimentin and TGFβ in human tumour tissueserial sections of frozen material were analysed immunohistochemicallyby the ABC method. The immunohistochemical analysis was carried out asdescribed by Heider et al., 1995. Anti-cytokeratin (clone MNF 116; DAKO,Denmark), anti-vimentin (clone V9; DAKO, Denmark) and a mixture ofanti-TGFβ1 and TGFβ2 (Santa Cruz, Calif.) were used as the primaryantibodies. The results of the staining were evaluated with a ZeissAxioskop microscope.

For the simultaneous detection of vimentin and cytokeratin in tissuesections, double fluorescence analysis was carried out. Anti-vimentin(clone V9; DAKO, Denmark) and an anti-cytokeratin rabbit serum were usedas primary antibodies, whilst Cy3-coupled anti-mouse IgG or FITC-coupledanti-rabbit IgG antibody were used as secondary antibodies. Thefluorescence was evaluated using a Zeiss Axiophot 2 microscope with theaid of a Leica Quantimed Q500 picture analysis system.

f) RNA in situ Hybridisation

For the RNA in situ hybridisation the frozen sections were fixed andextracted as described by Oft et al., 1993. For this the sections werefixed in 4% paraformaldehyde in PBS, washed twice in PBS, prehybridisedfor 2 hours and hybridised overnight with the appropriate S³⁵-labelledriboprobe at 52° C. in 50% formamide, 0.6 M NaCl. After washing understringent conditions (T_(m)−20° C.) the sections were immersed in KodakNTB liquid emulsion and illuminated for 2 weeks. The slides with thesections were counter-stained with haematoxylin/eosin and analysed underlight and dark field illumination using a Zeiss Axiophot microscopes.

In order to prepare the S³⁵-labelled riboprobes the hTGFβ1-cDNA (R&D)and the cDNA for neomycin phosphotransferase, excised from a suitableretroviral vector (Redmond et al., 1988), were cloned into the T₃-T₇expression plasmids (Bluescript II KS Stratagene) and transcribed invitro, in the presence of S³⁵-UTP for the antisense riboprobe and forthe sense control probe.

The non-radioactive in situ hybridisation for TGFβ in sections of humantumour tissue was carried out by means of digoxygenin-labelled probes.The probe (hTGFβ1, see previous paragraph) was labelled by means of theDIG-RNA Labelling Kit made by Boehringer Mannheim according to themanufacturer's instructions. For the hybridisation frozen sections (5-7μm) were fixed in 4% paraformaldehyde for 10 min, washed twice in PBSand subsequently acetylated for 10 min in 0.5% acetic anhydride. Afterwashing twice in PBS the sections were dehydrated in an ascendingalcohol series, air-dried and subsequently incubated for 30 min at 52°C. in a damp chamber. The hybridisation with the probe was carried outfor 4 to 6 h at 52° C. in a damp chamber. After hybridisation the slideswere washed for 2×10 min in 2×SSC at 52° C. and subsequently the boundprobe was made visible by means of anti-digoxygenin antibody accordingto the instructions of Boehringer Mannheim. The slides were brieflycounter-stained with haematoxylin, covered and evaluated with a ZeissAxioskop microscope.

g) Electron Microscopy

The cells grown in the collagen gels were pre-fixed for 10 min in 3%paraformaldehyde in 0.2 M HEPES pH 7.3 at room temperature. The cellswere further fixed on ice in 8% paraformaldehyde in 0.2 M HEPES pH 7.3for 30-60 min. For the immunocytochemistry the samples were dewatered inethanol at ever lower temperatures, embedded in Lowicryl HM20 or K4M andpolymerised at −35° C. by means of UV-light (Schwarz et al., 1993). Toinvestigate the ultrastructure the cells were post-fixed with 1% osmiumtetraoxide in PBS pH 7.2 for 1 h on ice, stained with 1% aqueous uranylacetate for 1 hour, dewatered in ethanol at room temperature and finallyembedded in Epon. For the immunocytochemistry ultrathin sections werestuck to cover glasses (Schwarz, 1994). After the blocking ofnon-specific antibody binding sites with 0.5% bovine serum albumin and0.2% gelatin in PBS the sections were incubated withrabbit-anti-Catenin-antibodies and subsequently with Cy3-labelledgoat-anti-rabbit IgG. The labelled sections were stained with4′,6-diamino-2-phenylindole (DAPI), to make the nuclei visible underimmunofluorescence microscopy.

h) Southern Blot Analysis

Total DNA of cells or tumour material was isolated and processed usingstandard methods (Maniatis et al., 1982). DNA, extracted from cellsbefore injection, from freshly excised tumour tissue (day 15 after theinjection) and from tumour tissue which had been recultivated in vitroin the presence of G418 for 5 days, was digested with the restrictionenzyme EcoRI (which cuts the retroviral vector only once), blotted ontoa Gene Screen Membrane and hybridised with the cDNAs, coding either forthe neomycin-phosphotransferase or for the v-Ha-ras gene.

i) Northern Blot Analysis

The Northern Blot analysis was carried out as described by Chomczynskiand Sacchi, 1987; as well as by Reichmann et al., 1992. Total RNA (10 μgper track) was placed on denaturing, formaldehyde-containing gels,blotted onto Gene Screen Membranes and hybridised with the entire codingregion of hTGFβ1-cDNA (R&D), which has sufficient homology with mTGFβ1,2 and 3 to recognise all three mouse-TGFβ-isoforms.

j) Semi-quantitative PCR

Total RNA from cells, grown on plastic, in collagen gels or fromtumours, was isolated and processed for the semi-quantitative PCR. ATGFβ1-specific fragment was amplified by means of RT-PCR undersemi-quantitative conditions, using -actin primers as the internalcontrol, as described by Leonard et al., 1993. For this the DNA wasdenatured at 94° C. for 1 min, the primers were annealed at 65° C. for 1min, and the polymerase reactions were continued at 72° C. for 1 min.The amplification was continued for 20 and 30 cycles. The TGFβ1-specificprimers TGGACCGCAA CAACGCCATC TATCCAGAAAA CC (forward) and TGGAGCTGAAGCAATAGTTG GTATCCAGGG CT (reverse) (Clontech Inc.) were used. Theresults of the PCR were quantitatively evaluated on an Image QuantPhospho-Imager. The values were standardised on the control product(-actin) and subsequently correlated with the value fromcontrol-3T3-fibroblast.

TGFβ1 was detected in tumour tissue by means of RT-PCR as brieflydescribed (Heider et al., 1996). The TGF β1-specific oligonucleotideGCCCTGGACACCAACTATT GCTTC was used as 5′-primer and the TGF β1-specificoligonucleotide TGCTCCACCTTGGGCTTGC was used as 3′-primer. Theamplification products were separated on a 2% ethidiumbromide-containing agarose gel and evaluated under UV-light by means ofa video camera (MWG Biotech).

k) Transient Transfection of a PAI-1-promoter-reporter Gene Construct

A PAI-1 promoter-reporter construct (reporter gene was luciferase;3TP-lux, Wrana et al., 1992) was transfected by lipofectaminetransfection (Gibco) according to the manufacturer's instructions intoCT26 cells or CT26-TβRII-dn cells. 8 hours after transfection TGFβ1 wasadded for 24 hours, while the controls were left without TGFβ. Then celllysates were prepared and the luciferase activity was measured in aBerthold Clinilumat as described by Wrana et al., 1992. In order todetermine the transfection efficiency all the cells were cotransfectedwith an CMV-β-Gal reporter gene construct and the luciferase activitiesobtained were standardised by the β-Gal fluorescence intensity and bythe protein concentration of the extract (Bradford).

1) Quantitative Determination of Soluble TGFβ by means of ELISA Assay

In order to determine the TGFβ concentrations by means of ELISA assay,EpH4, polarised EpRas and fibroblastoid EpRas cells, isolated fromtumours or converted in vitro with TGFβ, were washed five times with PBSto remove exogenous TGFβ and subsequently grown for 48 hours inserum-free DMEM. Then the cell culture supernatants were collected andthe TGFβ1 concentrations were determined by means of a commerciallyobtainable ELISA-Kit (Promega; G1230) according to the manufacturer'sinstructions.

m) Immunoblots

In order to determine TGFβ1 in the tissue culture supernatants, 2 mlserum-free cell supernatants were concentrated by ultrafiltration(Centricon 10, Amicon) down to a final volume of 0.1 ml. Theconcentrated supernatants were mixed with 5-times concentrated SDS-PAGEprobe buffer (without mercaptoethanol) and analysed by SDS-PAGE undernon-reducing conditions. Equal aliquots of protein (50 μg) weresubjected to SDS-polyacrylamide gel electrophoresis; the immunoblotanalysis was carried out as described by Hayman et al., 1993.

n) Chicken Embryo Heart Invasion Assay

This assay was carried out as described by Behrens et al., 1993. Inorder to be able to distinguish invasive donor cells clearly fromchicken heart cells, the test cells were charged with a vitalfluorescent dye before examination. For this the cells were incubatedfor 1 hour in a glucose-containing Hanks saline solution containing 10mM 5,6-carboxy-2′,7′-dichlorofluorescein diacetate-succinimidyl ester(Molecular Probes) and 0.2×10⁻⁶ M Pluronic F127. In this way thefluorescent dye is covalently bound to intracellular proteins withoutaffecting the viability or behaviour of the cells, determined by variousdifferentiation and proliferation assays. The labelled cells were grownfor 24 hours at high density, scraped off the plastic dish and broughtinto contact with pre-cultivated heart fragments of 9 day old chickenembryos on the surface of a soft agar layer. After 7 days' cultivationthe fragments with the adhering cells were collected, flash-frozen inliquid nitrogen, frozen sections were prepared, fixed inmethanol/acetone and the fluorescent cells were determined byepifluorescence microscopy (Axiophot, Zeiss).

o) Implantation of TGFβ1-charged Slow Release Pellets in Mice

In order to expose Ras-transformed breast epithelial cells to activatedTGFβ1 very early in tumour development, TGFβ1 -charged Slow ReleaseElvax Pellets and either EpRas-epithelial cells or normal EpH4H cellswere coinjected subcutaneously in mice. For control purposes pellets,charged only with BSA, were coinjected. The pellets were prepared andcharged according to the manufacturer's instructions.

Example 1

Ras Expressing Polarised Epithelial Cells Undergo EF Conversion DuringTumour Development

The tests carried out were suggested by the observation thatRas-transformed mouse-breast epithelial cells (EpRas cells) exhibit twocompletely different cell phenotypes. When they are grown on plasticsubstrates, these cells grow as ordered, dome-forming monolayers(hemicysts), indicating a polarised epithelial phenotype (FIG. 1A, B).After being injected into mice, however, these same polarised cellsformed tumours consisting of depolarised spindle-shaped cells with thecapacity for invasive growth (FIG. 1A, C). In order to obtain furtherfindings as to the mechanisms underlying this phenotypical plasticity,the cell conversion observed was examined in detail by a combination ofin vivo and in vitro experimental preparations. The cell clone EpH4 wasused for this, which is derived from a well characterised mouse-breastepithelial cell line (Reichmann et al., 1989; Reichmann et al., 1992;Strange et al., 1991). These cells exhibit a stable polarised epithelialphenotype (Reichmann et al., 1994).

When suitable retroviral vectors were used tumorigenic subclones of EpH4were formed by stable expression of the v-Ha-Ras-oncogene. After theexpression of v-Ha-Ras as been confirmed by Western Blot analysis, cellsfrom seven clones (referred to as EpRas-clones) were injectedsubcutaneously or directly into the mammary glands of Balb/c-mice.Tumours were formed regularly which were palpable 5-7 days after theinjection of the cells.

The phenotype of these converted tumour cells was compared with that ofthe original differentiated clones: before the injection all sevenEpRas-clones displayed the expected polarised phenotype (FIG. 1B andTable 1). By contrast, when cells were excised from the tumours andrecultivated in the presence of G418, only converted, fibroblastoidcells were obtained (FIG. 1C, Table 1). Although they still expressedcytokeratin to a certain extent, these cells had lost many of theirepithelial properties and acquired the expression of fibroblasticmarkers (FIG. 1C, Table 1). In order to demonstrate that thetumour-cells came from the EpH4 donor cells originally injected, as wellas to show that no rearrangement or reintegration of the Ras-containingretrovirus had taken place during the tumorigenesis and subsequentcultivation in vitro, the integration pattern of the retroviralconstructs was determined by Southern Blot analysis. For this EpRascells before injection, cells from a 15-day tumour and re-isolated cellsfrom a 30-day tumour were analysed. When using probes with specificityfor the neomycin resistance gene or the ras gene identical integrationpatterns were obtained in all three cell types (FIG. 1D).

The conversion of EpRas cells into fibroblastoid cells during tumourformation in mice is shown in FIG. 1.

FIG. 1A shows the principle of the strategy which was used in order tostudy EFC of Ras cells (7 different v-Ha-Ras expressing cell clones wereused) in vivo.

FIG. 1B: Before injection cells of the clone Ep5 exhibited the formationof domes on plastic and staining both on E-cadherin (FITC, greenfluorescence, appearing dark in the black and white representations),and also on cytokeratin (Texas-Red, red fluorescence). The commonstaining of both proteins on the periphery of the cell should be noted(yellow staining).

FIG. 1C: Ep5 cells, isolated from a tumour 28 days after the cellinjection. These cells display a fibroblastoid appearance and expresscytokeratin but no E-cadherin.

FIG. 1D: Southern Blot analysis. The EpRas-clone (Ep5), before injection(Ep5, plastic), removed from the tumour (Ep5, tumour), removed from thetumour and recultivated for 5 days in G418 (Ep5, ex tumour), shows thesame retroviral integration pattern (detected with aneomycin-phosphotransferase (NPT) probe).

Example 2

Timing of EF Conversion and Behaviour of Animal Donor and Receiver CellsDuring Tumorigenesis in vivo

Next, the stage of tumour development at which the subcutaneouslyinjected epithelial EpRas cells undergo EF conversion, if at all, wasexamined. Three days after the injection the EpRas cells formed clearlydefined nodules in which the cells expressed characteristiccytokeratins, but no vimentin (FIG. 2A). These epithelial cell noduleswere already encapsulated by stroma cells (FIG. 2A). Cells which grewout of these microtumours on plastic and in the presence of G418, stilldemonstrated epithelial properties. Seven days after the injection itwas observed that the solid cell aggregation of Ep-Ras cells wasbeginning to break up at the edge of the tumour and the epithelial cellswere mixed with vimentin-positive stroma cells at the periphery of themicrotumours (by inward migration of the stroma cells or outwardmigration of the donor cells). At this moment the donor cells stilldisplayed epithelial properties, both in the tumour and also afterisolation and in vitro cultivation in G418.

15 days after the injection three different cell types could bedistinguished (FIG. 2C): about 20% of the tumour cells were greenstained vimentin-positive stromal cells. Another 20% expressed onlycytokeratins, indicating EpRas cells which have retained the epithelialphenotype. The majority (50-60%) of the tumour mass, however, consistedof cells which co-expressed cytokeratin and vimentin. These cells are inall probability converted or converting EpRas cells. Both the epithelialand also the converted fibroblastoid cells were also obtained after G418selection. Finally, the epithelial part could no longer be detected,either in situ, or on plastic, in five week old, fully developedtumours. By contrast parental EpH4 cells never formed tumours. When theywere injected subcutaneously, the EpH4 cells developed into layers ofepithelial cells which sometimes formed lumina and cytokeratins, butexpressed no vimentin (FIG. 2D). After a fairly long time these cellsnecrotised and were reabsorbed by the surrounding stroma.

In order to clearly identify the originally injected donor cells at thethree different tumour stages, in situ hybridisation was carried out onthe neomycin resistance gene. These experiments showed that allcytokeratin expressing cells originated from donor cells. The frequencyof donor cells relative to the stroma cells of the receiver animalsincreased with the size of the tumour and was greatest in fullydeveloped tumours (FIGS. 2E, F, G, H).

All in all, these data show that both the Ras expressing cells and alsothe epithelial control cells in vivo initially have an epithelialphenotype. As the development of the Ras cell tumours progresses theRas-transformed cells progressively acquire fibroblastoid properties. Bycontrast the non-tumorigenic parental cells stably retain theirepithelial properties until they die.

FIG. 2 shows the timing of the epithelial/mesenchymal conversion (EFC)during tumour development and demonstrates the fate of donor andreceiver cells during this process.

Differently treated frozen sections of EpRas-tumours (clone Ep2) areshown, which were prepared on day 3 (FIGS. 2A, E), on day 7 (FIGS. 2B,F), on day 15 (FIGS. 2C, G) and on day 28 (FIG. 2H) after the injection.The cell structures formed by non-tumorigenic EpH4 cells 15 days afterthe injection are shown in FIG. 2D. The sections were examined byimmunofluorescence (FIGS. 2A-D) and in situ hybridisation (FIGS. 2E-H).The sections were double-stained with antibodies against a 46 kDacytokeratin (Texas-Red, red fluorescence) and vimentin (FITC, greenfluorescence). It was noted that in 3-day-old tumours the injectedepithelial cells (stained red) and the mesenchymal cells of the host(stained green) are clearly separate. In 15-day-old tumours largenumbers of cytokeratin/vimentin-double-positive cells are visible(yellow-stained cells). These cells have undergone EFC. RNA-in situhybridisation using a neomycin-phosphotransferase-probe confirms thedonor origin of the tumour cells and shows the increasing density of thetumour cells after EFC (FIGS. 2E-H).

Example 3

TGFβ1 Induces in vitro EF Conversion in Ras Expressing, but not inNormal Epithelial Cells

In order to identify the mechanism underlying the EF conversion, anexperimental system was used with which EF conversion can be induced invitro under defined and physiologically relevant conditions. For thispurpose normal EpH4 cells or Ras-transformed subclones of these cells(Ep-Ras clones) were grown in reconstituted collagen type I gels, usingserum-free medium. These conditions made it possible to add definedpolypeptide growth factors and hormones which are known to be involvedin the modulation of the epithelial phenotype. In gels of this kind,normal EpH4 cells developed into organ-like gland channels (tubuli)which frequently terminated in club-shaped hollow swellings. Thesestructures looked very similar to the end buds of the developing mammarygland which are formed by primary breast epithelial cells both incollagen gels and also in vivo (FIG. 3A). These structures could beinduced to produce milk proteins efficiently by the addition oflactogenic hormones. When these cells were isolated from the gel andgrown on tissue culture plastic, they formed the expected regularepithelial monolayers, which formed recognisable domes, an indicationthat these cells are able to polarise efficiently (FIG. 3, right-handTable).

Surprisingly, the EpRas-clones in these serum-free collagen gels alsoexhibited considerable lumen formation. The lumina were visible as earlyas 2-3 days after seeding. Thereafter more than 95% of these structuresdeveloped relatively large cystic cavities (FIG. 3B, left-hand andcentre Table) which resembled the alveoli of the fully developedmilk-producing mammary gland. On plastic these cells in turn formedregular epithelial monolayers with domes, and thus exhibited the sameepithelial properties as the non-tumorigenic starting cells (FIG. 3B,right-hand Table).

The same EpRas cells behaved completely differently, however, when theywere cultivated in 10% foetal calf serum (FCS). Under these conditionsthey formed elongated, multi-cellular and invasively growing strings ofcells which never showed any lumen formation. These strings consisted ofnon-polarised cells which had lost many epithelial properties (FIG. 3Cand FIG. 4) and behave in a strikingly similar manner to the ex vivofibroblastoid tumour cells. These findings indicated that a factorcontained in the FCS, co-operating with the activatedHa-Ras-oncoprotein, brings about the conversion of the epithelial EpRascells into fibroblastoid cells.

In order to identify this factor or these factors, a number of growthfactors (TGFβ, heregulin, scatter-factor/hepatocyte growth factor,acidic and basic FGF, PDGF and TGFβ1) were added to the Ras-transformedcells grown in collagen gels. Surprisingly, TGFβ1 was the only factorwhich showed striking and long-lasting effects on EpRas cells. WhenTGFβ1 was added, these cells grew into elongated, branching strings ofcells similar to those induced by FCS. On tissue culture plastic thesecells exhibited a clear, fibroblastoid phenotype (FIG. 3D). In pH4control cells and other non-tumorigenic breast epithelial-cell clones,by contrast, TGFβ1 was not able to induce EF conversion.

In order to examine whether the activity in the serum which promotes EFconversion is actually TGFβ1, cultures which contained 5t FCS wereincubated with TGFβ1 neutralising antibodies. Under these conditionsEpRas cells in turn formed cystic cavities very similar to those shownin FIG. 3B. Thus, the cell-converting activity present in FCS wasidentified as TGFβ1 and it was shown that TGFβ1 is the only or at leastthe predominant activity in FCS which can induce EF conversion.

Other ultrastructure and immunohistochemical analyses showed that mostof the cystic structures consisted of a monolayer of polarised cells(FIG. 4A). These cells abundantly formed microvilli at their apicaldomain (the one facing the lumen), indicating a polarised organisationof the cells (FIG. 4A). Moreover, different types ofepithelial-cell-typical cell-to-cell contact structures, i.e. tightjunctions, characterised by the protein ZO-1, desmosomes (FIG. 4A) andthe cell adhesion molecule E-cadherin typical of so-called “adherensjunctions” (FIG. 4B) could be detected by their typical lateral orbasolateral positions. Similarly, the protein β-catenin associated withE-cadherin showed basolateral localisation in most of the cells (FIG.4C).

By contrast, the string-like cell structures induced by TGFβ1 consistedof loosely adhering spindle-shaped cells (FIG. 4D, inset picture). Noneof the epithelial marker proteins and ultrastructurally recognisablecontact structures mentioned could be detected (FIG. 4D and Table 1),with the exception of a low, non-polarised expression of E-cadherin(FIG. 4E). The expression of β-catenin was greatly reduced and locatedchiefly in the cytoplasm (FIG. 4F). Moreover, these cells expressed theexpected mesenchymal markers (Table 1).

These results show that Ras-transformed mouse-breast epithelial cellsexhibit exceptional plasticity in the phenotype, which ranges fromepithelially polarised cells organised into ordered epithelia tofibroblastoid, migratory and invasively growing cells.

FIG. 3 shows the destruction of lumen formation and epithelial polarityby serum and TGFβ1.

Non-tumorigenic EpH4 cells (FIG. 3A) or tumorigenic EpRas cells (cloneEp5, FIGS. 3B-D) were grown in collagen type I matrices. Themacroscopically visible structures were photographed 8 days afterplating out at low and high magnifications (left-hand and middle Table).Cells isolated from the gels and grown on tissue culture plastic areshown in the right-hand Tables.

FIG. 3A: Ep4H cells form channels and swellings resembling end-buds inserum-free collagen gels. On plastic these cells formed a regularepithelial monolayer and domes (hemicysts).

FIG. 3B: In serum-free collagen gels, wide channels and alveoli-likecysts are formed by EpRas cells.

FIG. 3C: Addition of 10% FCS causes the cells to form invasively growingirregular strings of cells without a lumen. On plastic these cells aresimilar to fibroblasts and are spindle-shaped.

FIG. 3D: TGFβ1 on its own (5 ng/ml) causes EpRas cells to grow intoinvasive strings of cells similar to those induced by FCS.

FIG. 4 shows the breakdown of epithelial cell polarity inRas-transformed breast epithelial cells after incubation with TGFβ1.

Alveoli-like cysts, formed by EpRas cells (clone Ep6) in serum-freecollagen gels (FIGS. 4A-C), and disordered strings of cells, formed bythe same cells after treatment with TGFβ1 (FIGS. 4D-F), were analysedfor their epithelial organisation and formation of cell polarity.Sections through individual structures were photographed at high or lowmagnifications(inset pictures).

FIG. 4A: Transmission electron microscopy showed that the cysts obtainedin the absence of TGFβ1 consisted of a monolayer of morphologicallypolarised cells which finally comprise the microvilli in their apicaldomain, facing the lumen (FIG. 4D). The inset picture shows a monolayercyst of this kind at low magnification. By contrast, the strings ofcells induced in the presence of TGFβ1 consist of loosely adhering cellswithout microvilli, desmosomes or tight junctions.

FIGS. 4B, E: frozen sections through an alveolar cyst which wereimmunostained with an antibody against the cell adhesion moleculeE-cadherin, showed clear basolateral localisation of the E-cadherins inmost of the cells. In the TGFβ1-induced strings of cells, E-cadherin isreduced in its expression and is expressed over the entire surface ofthe fibroblastoid cells.

FIGS. 4C, F: These show Lowicryl sections through structures similar tothose shown in FIGS. 4B and E, immunostained with ananti-β-catenin-antibody. The basolateral expression of β-catenin in mostof the cells of the cyst (FIG. 4C) and the significantly reducedβ-catenin expression which is now localised predominantly in thecytoplasm should be noted (FIG. 4F).

Example 4

Fibroblastoid EpRas Cells are Invasive

EpRas cells which had undergone EFC showed signs of invasive behaviourin collagen gels. In order to obtain definitive proof of this invasiveproperty, the chicken embryo heart invasion assays were used, therelevance of which to in vivo metastasisation has already beendocumented in detail (Mareel et al., 1979; Mareel, 1983). In this assaythe migration of cells into embryo heart fragments was examined (FIG.5A). In order to identify the penetrating cells clearly, they werelabelled with a fluorescent dye(carboxy-dichloro-fluorescein-diacetate). During the incubation periodof seven days no parental EpH4 cells migrated into the chicken hearttissue (FIGS. 5A, B). In three different, fully-polarised Ep-Ras-clones,only a vanishingly small proportion of the cells were capable ofmigrating into the heart tissue (FIG. 5C). The few cells which migratedin were strongly stained with a vimentin antibody, but not with ananti-E-cadherin antibody. This confirms their conversion into afibroblastoid phenotype, which is not surprising as the co-culturescontained serum. In contrast to the epithelial cells the fibroblastoidcells which had been obtained from tumours (“ex-Tu cells”), or cellswhich had been induced to EFC by the use of TGFβ1 in vitro, migratedinto the heart muscle tissue in large numbers and relatively fast (FIG.5D). These results show that EpRas cells are highly invasive afterundergoing EFC, while non-converted epithelial cells exhibit only slightinvasivity.

FIG. 5 shows the high invasivity of fibroblastoid EpRas cells in thechicken embryo heart invasion assay.

In vivo fluorescence-labelled cells were co-cultivated with chickenembryo heart fragments in order to test their invasivity, and sectionsthrough the fragments were examined histologically 7 days later. Thenon-tumorigenic epithelial starting cells (EpH4 cells) did not migrateinto the heart fragments (FIGS. 5A, B), and non-converted epithelialEpRas cells showed only slight invasivity (FIG. 5C). By contrast theconverted, fibroblastoid cells obtained after TGFβ1 treatment werecapable of migrating efficiently into the heart fragments (FIG. 5D).

Example 5

TGFβ1 Maintains the Fibroblastoid Phenotype of Converted EpRas Cellsthrough an Autocrine Loop

After it had been shown that TGFβ1 converts Ras-transformed epithelialcells into fibroblastoid cells, the question arose as to whether TGFβ1is also involved in maintaining this phenotype. A possible explanationfor the relative stability of the fibroblastoid phenotype (e.g. incultures on plastic) was the autocrine production of fairly largeamounts of TGFβ1 by the converted cells themselves. In order to settlethis question, fibroblastoid EpRas cells were cultivated in extremelylow concentrations in 1% FCS (in order to minimise theTGFβ1-concentration in the medium). Under these conditions individualcells grow into clearly spatially separated clones. As shown in FIGS. 6Aand B, the clones obtained soon after plating out and consistinginitially of a few cells had a fibroblastoid morphology at first. As thenumber of cell clones increased, the cells in the overwhelming majorityof the clones gradually changed into cells with an epithelial phenotype(FIGS. 6A to D). This reversion was substantially complete 10 days afterplating out (FIG. 6D).

In order to suppress the effects of autocrinally produced TGFβ1completely and thereby definitively demonstrate that TGFβ1 is reallynecessary for maintaining the EF conversion, fibroblastoid cells(ex-tumour cells) isolated from a tumour were grown in the presence orabsence of TGFβ1 neutralising antibodies in collagen gels. In theabsence of the antibodies the fibroblastoid tumour cells formed theexpected thin, invasively growing strings of cells (FIG. 6E). The samecells, however, no longer grew invasively and developed into cysticstructures consisting of an epithelial monolayer when they were treatedfor eight days with the neutralising antibodies (FIG. 6F).

Finally the amounts of TGFβ1-mRNA expressed in the cells and the TGFβ1-protein released into the culture medium were determined. Threedifferent EpRas clones as well as the parental EpH4 clone were grown forfive days in collagen gels. When they were treated with 5 ng/ml TGFβ1,the EpRas-clones underwent EFC, while the similarly treated,non-transformed EpH4 cells retained their epithelial phenotype. Analysisof these cells by semi-quantitative PCR (FIG. 7A) or by means ofimmunoblot (FIG. 7B) showed that the fibroblastoid cells induced byTGFβ1 produced quantities of TGFβ1-mRNA which were comparable with thoseof control fibroblasts (FIG. 7A). This also applied to EpRas cells whichhad changed into fibroblastoid cells in tumours. By contrast, parentalEpH4 cells and epithelial EpRas cells produced no or only small amountsof TGFβ1-mRNA (FIG. 7A). At the protein level essentially the sameresults were obtained when serum-free culture supernatants were analysedby ELISA and Western blot (FIG. 7B).

FIG. 6 shows that TGFβ1 maintains the fibroblastoid phenotype ofconverted EpRas cells through an autocrine loop.

FIGS. 6A-D: clones from fibroblastoid cells isolated from a tumour(ex-tumour cells) gradually change into clones consisting of epithelialcells. In order to produce the clones 500 cells per 100 mm dish weresown in medium containing 1% FCS. The medium was changed daily in orderto dilute any autocrine factors. The same typical cell clone wasphotographed on day 1 (A), day 3 (B), day 5 (C) and day 10 (D) afterplating out. The gradual transformation of the fibroblastoid cells intocells with an epithelial morphology is clearly visible.

FIGS. 6E, F: fibroblastoid EpRas cells isolated from a tumour wereselected for 5 days in G418 (in order to eliminate any cells originatingfrom the receiver animal) and subsequently seeded into serum-freecollagen gels. This was carried out either in the absence (E) or in thepresence (F) of TGFβ1 neutralising antibodies. It can be seen that inthe presence of a TGFβ1 neutralising antibody the tumour cells developinto lumen-shaped structures, whilst in the absence of the antibody theyform the expected disordered strings of cells.

FIG. 7 shows that converted EpRas cells produce high concentrations ofTGFβ1.

FIG. 7A: RNA from non-converted (epithelial) and converted(fibroblastoid) EpRas cells (clone Ep5) and also from non-tumorigenicEpH4 cells and NIH-3T3-fibroblasts (ATCC CRL 1658) was used forsemi-quantitative PCR analysis. The significant increase in TGFβ1-mRNAin the fibroblastoid cells should be noted. The TGFβ1 expression isrecorded as a percentage of the values obtained with NIH-3T3 cells.

FIG. 7B: Similar results were obtained when the TGFβ1-concentrations incell culture supernatants were analysed by Western Blot and ELISA (thenumbers above the Western-Blot gel traces show the quantities of TGFβ1in ng TGFβ1/ml determined in the ELISA). The data shown in FIG. 7B wereconfirmed with two other EpRas-clones (Ep2 and Ep6).

In all, these results indicate the major role of TGFβ1 not only ininducing EFC, but also in maintaining the fibroblastoid phenotype.

Example 6

Finally tests were carried out to determine whether TGFβ1 is actuallyexpressed in EpRas tumours and whether TGFβ1 added experimentally invivo can also bring about EFC and invasivity of the cells. Tumoursgrowing from injected EpRas cells were examined for the expression ofTGFβ1, 4 and 15 days after the injection of the cells, by RNA in situhybridisation and immunohistochemistry. Just 4 days after the injectionof the cells increased concentrations of TGFβ1-mRNA were detected at theouter edge of the nodes formed by the EpRas cells (FIG. 8A). Theco-expression of TGFβ1 and neomycin phosphotransferase (NPT, which isexpressed exclusively by the Ras-transformed donor cells) shown up bydouble immunofluorescence showed that the great majority of the donorcells (characterised by the red staining on NPT) produced no TGFβ1(green staining) at this stage of the tumour development. On the otherhand, cells of the surrounding tumour stroma originating from thereceiver animal and non-epithelial in origin were distinctly positivefor TGFβ1 (FIG. 8B). By contrast tumours which had been removed 28 daysafter the injection showed a relatively high and uniform expression ofTGFβ1-mRNA over the entire tumour region (FIG. 8C). In these tumours itwas found that the injected EpRas cells themselves produced TGFβ1because they could be stained with antibodies against both NPT andTGFβ1; they displayed a yellow staining (FIG. 8D). Remarkably, most ofthe cells which produced TGFβ1 showed a reduced expression ofcytokeratin, whereas the majority of the cells with high cytokeratinexpression could not be stained with antibodies against TGFβ1. This isfurther proof that the converted cells are actually whose which alsoproduce TGFβ in the animal at advanced stages of the tumour.

These results show that host cells which surround the tumour tissue areable to initiate cell conversion. The converted tumour cells in turnthemselves produce TGFβ, thus speeding up cell conversion andsubsequently the invasion processes.

In order to prove this directly, Slow Release Pellets charged withrecombinant human TGFβ1 were applied close to the injected EpRas cells.The same TGFβ1 pellets, combined with non-tumorigenic EpH4 cells, wereused as controls. Surprisingly, EpRas cells located close to a TGFβ1pellet were converted into irregularly shaped cells just 4 days afterthe injection and exhibited extensive migration into the surroundinghost tissue. Surprisingly, even at this early stage, many of these cellswere positive for vimentin (FIG. 8F). By contrast, identical EpRas cellswhich had been injected in the absence of exogenous TGFβ1 formed smoothhomogeneous nodes of vimentin-negative cells forming close cell contacts(FIG. 8E). As expected, TGFβ1 pellets located close to EpH4 cells couldnot noticeably influence the phenotype of these non-tumorigenic cells.These in vivo data conform to the results obtained in vitro and lead oneto conclude that TGFβ1 has a key role in regulating the plasticity andinvasivity of tumour cells.

FIG. 8 shows how TGFβ1 in experimentally induced tumours triggers thetransition from the epithelial to the fibroblastoid state as well as theinvasivity of the cells:

FIGS. 8A-D: frozen sections of tumour stages on day 4 (A, B) and day 15(C, D). The RNA in situ hybridisation shows that on day 4 the TGFβ1expression is taking place in the outer periphery of the tumour (A), buton day 15 (C) it is occurring throughout the tumour. Arrow headsindicate the boundary between tumour and surrounding stroma.

FIGS. 8B, D: frozen sections were stained with an anti-TGFβ1-antibody(green fluorescence) and an anti-neomycin phosphotransferase-antibody,which recognises the donor cells (red fluorescence). The smallerdiagrams show extracts at higher magnifications. It should be pointedout that at early stages of the tumour TGFβ1 is produced exclusively bythe stroma around the tumour (B). By contrast, in 15 day-old tumours,TGFβ is also expressed in many donor cells within the tumour tissue (D,yellow fluorescence).

FIGS. 8E, F: Epithelial EpRas cells were injected subcutaneously intonude mice without (E) or together with 3-Elvax Slow Release Pelletscharged with recombinant (active) TGFβ1 (F). Frozen sections obtainedfrom 4 day old tumours were double-stained with antibodies againstcytokeratin (red) and vimentin (green). What is noticeable is thedramatic migration of cells into the surrounding tissue induced in thevicinity of the TGFβ1 -releasing pellets (white circle).

Example 7

Effect of TGFβ1 on Normal Breast Epithelial Cells: Control of Milk DuctMorphogenesis by Regulating Cell Growth, Cell Polarisation and Apoptosis

Since the TGFβ-super-family of polypeptide factors is involved primarilyin morphogenetic processes during embryo development, the role of TGFβ1in normal mammary gland development was also examined within the scopeof the present invention. For this purpose normal breast epithelialcells of the cell line EpH4 were sown in serum-free collagen gels.Unlike in the experiments in Example 3, the serum needed during sowingfor the collagen gel to set and washed out one day later was speciallyselected for a low content of TGFβ1. Under these conditions the in vitroorganogenesis was completely inhibited, and no tubular structures wereformed (FIG. 10A). When low concentrations of TGFβ1 (0.1 ng/ml) wereadded, the cells were able to proliferate and form atypical structureswhich generally lacked lumina (FIG. 10B). Further investigations showed,however, that these structures expressed ZO-1, a tight-junction protein,on the inside. Thus, these structures bore some resemblance to the endbuds of the developing mammary gland.

By contrast, higher concentrations of TGFβ1 (>0.25 ng/ml), caused thenormal epithelial cells to stop growing and die off by programmed celldeath (apoptosis) (FIG. 5C). This is an important difference between thenormal epithelial cells and the Ha-Ras-containing cells. Whereas thelatter are not induced into apoptosis and undergo EFC without exceptioneven concentrations of TGFβ1 which are 20 times higher (5 ng/ml), theTGFβ1 concentration which regulates the morphogenetic processes innormal breast epithelial cells is strictly laid down. Possibly, aberrantmorphogenesis caused by excessively high TGFβ1 concentrations isprevented by the fact that growth inhibition and apoptosis are inducedin the cells instead.

The fact that it was not possible to induce fully differentiated tubularstructures consisting of polarised cells with low concentrations ofTGFβ1, might be due to suboptimal culture conditions. On the other handthe complete organogenesis of tubular structures might depend on TGFβ1only being present during certain phases of the organ development. Inorder to examine this, the cells were treated with 0.1 ng/ml TGFβ1, asdescribed above, until structures had formed, then TGFβ1 was washed outof the collagen gel. Surprisingly, the atypical structures thenreorganised themselves without lumina and formed well-shaped tubularstructures with typical lumina (FIG. 10D, transient TGFβ1). Theseresults lead one to conclude (i) that TGFβ1 is absolutely necessary forin vitro organogenesis, (ii) that the concentration is critical, withhigher concentrations leading to apoptosis, and (iii) that TGFβ1 onlyhas to act on the cells during certain phases of the organ development.This normal function of TGFβ1 in the development of breast epithelialcells is completely changed in the Ras-transformed cells, with TGFβ herecausing an extremely abnormal form of tissue reorganisation which causesa transition from the epithelial to the fibroblastoid state (EFC) over awide range of concentrations.

The next step was then to look for indications that TGFβ1, analogouslyto these in vitro findings, also controls the morphogenesis andprogrammed cell death of mammary gland epithelia in vivo. For this,mammary glands in mice during puberty were subjected to histologicalanalysis combined with in situ hybridisation using a probe againstTGFβ1.

During this phase the virginal mammary glands grow into the surroundingfatty tissue (fat pad). Growth, differentiation and morphogenesis of themammary gland produced start from a structure which is termed the endbud and contains undifferentiated, not yet fully polarised epithelialcells. Sections through the end bud (where the proliferation andsubsequent organogenesis, such as e.g. the branching off of the milkducts, take place) were compared with sections through fullydifferentiated ducts of the gland (cf. the schematic diagram in FIG. 11,centre). Whereas TGFβ1 is produced in the mesenchymal stroma whichsurrounds the growing end buds (FIG. 11, left-hand Table), no suchproduction of TGFβ1 was seen in the stroma cells surrounding an alreadydifferentiated gland duct (right-hand Table). These findings largelycorrespond to the in vitro data, in which temporary, pulsed treatment ofthe breast epithelial cells with TGFβ1 was necessary for the tubularmorphogenesis.

Similarly, there was an indication that TGFβ also regulates theprogrammed cell death (apoptosis) of breast epithelial cells in vivo.During reversion of the mammary gland after ablactation the alveolarcells undergo mass apoptosis, whereas the cells in the gland ductssurvive and are retained. New growth of the mammary glands duringanother pregnancy starts from these cells. In order to examine thepossible involvement of TGFβ1 in this process, frozen sections throughthe dying alveolar zone of a mammary gland and through an adjacent glandduct region were prepared three days after the end of lactation (FIG.12, representation in the centre). In the region which had justundergone apoptosis, the mesenchymal cells surrounding the dying alveoliexpressed high concentrations of TGFβ1 (FIG. 12, left-hand Table), whilethe mesenchymal cells surrounding the surviving ductal structuresexpressed no TGFβ1 (FIG. 12, right-hand Tables). In both cases it isprobable that crosstalk takes place between the epithelial cells and theTGFβ1 production induced in the mesenchyme.

FIG. 10 shows that a low concentration of TGFβ1 controls the in vitromorphogenesis of normal mammary gland epithelial cells, particularlywhen the factor is given transiently. Higher concentrations of TGFβ1cause apoptosis in the same cells.

Normal EpRas cells were sown in collagen gels, using a foetal calf serumselected for a particularly low TGFβ1 content during the sowing. Underthese conditions the cells do not form any tubular structures (FIG.10A). In the presence of 0.1 ng/ml of TGFβ1 the cells form branchedstructures, but these lack lumina (FIG. 10B). If the TGFβ1 is removedfrom cultures with such structures on day 7 by washing, the cells formdistinct hollow structures (FIG. 10D). Higher concentrations of TGFβ1cause cell death (apoptosis, FIG. 10C, lower magnification on the left,higher magnification on the right).

FIG. 11: shows the in vivo expression of TGFβ1 during the formation ofthe normal mammary gland during puberty (day 25).

Frozen sections through end buds of a virginal mammary gland (left-handpanels) or through already formed gland ducts (right-hand panels) wereprepared as shown in the central diagram. Successive sections in aseries of sections were subjected to RNA in situ hybridisation for TGFβ1mRNA (upper panels) or histologically stained. It is clearly apparentthat mesenchymal cells which surround the end bud strongly express TGFβ1(left-hand panels), whereas in cells which surround the differentiatedgland ducts, there is no detectable TGFβ1 expression.

FIG. 12 shows the in vivo expression of TGFβ1 in the breakdown of thefully developed mammary gland after ablactation.

Young mice were taken away from their nursing mothers, thus triggeringthe reversion of the fully developed mammary gland. 3 days later frozensections were taken through the dying areas of the mammary gland(left-hand panels) as well as through the gland ducts unaffected by theapoptosis (right-hand panels) (cf. the diagram in the centre of theFigure) The sections were then examined for TGFβ1 expression, asdescribed in the legend to FIG. 11. Whereas TGFβ1 producing cells areclearly detectable in the area surrounding the dying alveoli (left-handpanels) there are none around the surviving gland ducts.

Example 8

Coexpression of Vimentin and Cytokeratins in Human Tumour Tissue.Expression of TGFβ by Human Primary Tumours

In the previous Examples a well characterised cell model, namelyRas-transformed breast epithelial cells from the mouse, was used. It wasthus very important to assess how far the results obtained with thismodel system as to the activity of the TGFβ receptor on the phenotypicalplasticity and invasivity of epithelial tumour cells applies to humancarcinomas. For this purpose 31 kidney cell carcinomas and 64 breasttumours of different degrees of malignancy were examined byimmunohistochemistry. Firstly, corresponding histological sectionsthrough such tumours were double-labelled with antibodies againstgeneral epithelial cytokeratins and antibodies against the mesenchymalmarker vimentin. Tumour cells which coexpress both markers have probablyundergone EFC. Secondly, adjacent sections from the breast tumours werelabelled with antibodies against human TGFβ1 and TGFβ2, in order to findout whether the tumour cells also produce TGFβ.

As shown in the following Table, 74% of the kidney cell carcinomasexpressed both cytokeratin and also vimentin in the degenerateepithelial tumour cells. As expected, the fibroblastoid cells of thetumour stroma expressed only vimentin, but no cytokeratin. In the breastcarcinoma cells the percentage of tumours which coexpressed cytokeratinand vimentin was smaller, namely between 24 and 27%.

The results of the histochemical analysis of the same breast tumours forthe expression of TGFβ were even clearer. Here, all the tumours testedshowed clear staining of the tumour cells for TGFβ (Table). The tumourstroma was stained more weakly or not at all for most of the tumours.The specificity of the staining was also clear from the staining ofnormal tissue; on the skin, for example, as expected, only the basalcell layers of the keratinocytes were positive. As is also shown in theTable, the results of the histochemical staining were also fullyconfirmed by in-situ hybridisation for TGFβ as well as by RT-PCR.

These results show that for a significant proportion of the humantumours investigated, there were clear indications, in two ways, thatthe tumour cells corresponding to the model in FIG. 9 had both undergoneEFC and had also highly regulated the production of TGFβ.

The Table shows that human kidney cell and breast carcinomas coexpresscytokeratin and vimentin. This is a clear indication that EFC has takenplace. Similarly, all the tumours investigated produce TGFβ.

The upper part of Table (A) shows the results of the staining forcytokeratin and vimentin on frozen sections of the types of tumourspecified. The lower part (B) shows the results of staining for TGFβ onsections through the same breast tumours. Footnotes give the results ofcontrol experiments for the TGFβ expression (by RT-PCR) and theexpression of TGFβ in the tumour stroma.

A Coexpression of Vimentin and Basal Cytokeratins

Number of tumours Number of with Type of tumours vim./cytok. tumourSubtype analysed coexpr. kidney 31 23/31 (74%) cell carcinomas (RCC)breast 64 18/64 (28%) tumours fibroadenoma (FA) 3 2/3 (66%) invasiveductal ca. 34 8/34 (24%) (IDC) invasive lobular ca. 26 7/26 (27%) (ILC)inv. ductal-lobular ca. 1 1/1 (IDLC)

B Expression of TGFβ-1/2

In situ Type of Antibody hybridi- tumour Subtype staining sation breastfibroadenoma (FA) 3/3 (100%) tumours invasive ductal ca. 33/33 (100%)13/13 (100%) (IDC) invasive lobular ca. 23/23 (100%) 9/9 (100%) (ILC)inv. ductal-lobular ca. 1/1 (100%) (IDLC)

Additional Analyses

1. RT-PCR of IDC and ILC: 11 cases positive with Ab are also positivewith RT-PCR

2. expression in the tumour stroma: antibody: in 35/61 cases, slightstaining In situ hybridisation: in 3/22 cases

Example 9

Neutralising Antibodies Against TGFβ Prevent Invasive Growth of HumanTumour Cell Lines in Collagen Gel

In Example 8 histochemical examination of sections through tumour tissueprovided evidence that the hypotheses reached with the model systemregarding TGFβ-induced EFC and the subsequent autocrine production ofTGFβ also apply to many human tumours. In order to obtain more directevidence of this, experiments were caried out to determine whether humantumour cells which grow invasively in the collagen gel can be convertedinto non-invasively growing cells by the administration ofTGFβ-neutralising antibodies. The kidney carcinoma cell line MZ 1795 andthe nasopharyngeal-carcinoma line KB were-used. The cells of both linesgrew in collagen gels containing 5% FCS without TGFβ-antibodies or afterthe addition of TGFβ to form networks and strings of fibroblastoid cells(FIG. 13, right-hand panels). In the presence of TGFβ neutralisingantibodies (cf. Example 5, FIG. 6), on the other hand, the cells formedcompact clumps without any reference to invasive growth (FIG. 13,left-hand panels).

FIG. 13 shows that TGFβ neutralising antibodies prevent the invasivegrowth of human tumour cell lines in collagen gel.

MZ 1795 cells and KB cells were sown in serum-free collagen gels towhich was added either 2% serum or 5 ng/ml of TGFβ (+TGFβ, right-handpanels) or to which a mixture of different antibodies against TGFβ(−TGFβ, left-hand panels; cf. Example 5, FIG. 6) was added. After 10days microphotographs of the cells in the collagen gels were prepared.Whereas both the MZ 1795 cells (top panels, higher magnification, bottompanels; summaries, lower magnification) and also the KB cells (lowerpanels) grow en masse into the collagen gel, when TGFβ is present(right-hand panels), the same cells in the presence of TGFβ-neutralisingantibodies form compact clumps without any cells growing out (left-handpanels).

Example 10

The Expression of a Dominant-negative TGFβ Receptor Prevents EFConversion and Slows Down Tumour Growth of Ras-transformed BreastEpithelial Cells

The most direct proof of the presumed mechanism of activity (proof ofprinciple) for the activity of TGFβ receptor inhibitors in inhibitingtumour progression consists in demonstrating this activity directly inthe tumour-bearing animal. This was not possible within the scope ofthese Examples with the TGFβ-neutralising antibodies used in Vitro, asthe large amounts of antibody. needed for in vivo tests of this kindwere not available. An alternative approach was therefore adopted.

There is a “kinase-dead” mutant of the human TGFβ receptor type II(TβRII-dn), which also acts as a dominant-negative receptor (i.e. onethat switches off the function of the wild-type receptors). A cDNA ofthis TβRII-dn was expressed in Ras-transformed EpH4 cells (Ep-Ras) withthe aid of retroviral vectors. The clones obtained grew very slowly andrequired medium with a high (20%) serum content, in order to be capableof expansion. After injection into nude mice these cells had formedeither no tumours at all or only small tumours (FIG. 14, top) up to thetime when the mice injected with control cells (Ep-Ras) had to be killedbecause of their excessively large tumours.

The tumour cells were isolated from the slowest-growing Ep-Ras-TβRII-dntumour as well as from a control tumour induced by Ep-Ras cells andcultivated (cf. Example 1). Whilst the cells of the control tumourexhibited the expected fibroblastoid morphology (bottom of FIG. 14,left-hand Table) the tumour cells isolated from the slow-growingEp-Ras−TβRII-dn exhibited a distinctly epitheloid morphology (bottom ofFIG. 14, right-hand Table). This shows that the EF conversion occurringduring tumour formation by Ep-Ras cells is inhibited by the expressionof TβRII-dn and that this leads to a slowing down of tumour growth.

FIG. 14 shows that TβRII-dn expressing, Ras-transformed breastepithelial cells (Ep-Ras−TβRII-dn) exhibit slower growth in the animaland the cells isolated from these tumours have not undergone any EFconversion.

Four different clones of Ep-Ras−TβRII-dn cells as well as an Ep-Rascontrol clone were each injected subcutaneously into 3 nude mice (1×10⁶cells per animal). After 3 weeks the tumours were excised and weighed.The diagram in the upper part of FIG. 14 gives the mean values of thetumour weights obtained. The tumour cells from an Ep-Ras−TβRII-dntumour, obtained from the slowest tumour-forming Ep-Ras−TβRII-dn clone,and an Ep-Ras control tumour were cultured, selected in G418 (cf.Example 1) and photographed after 10 days under phase contrast. Thebottom left-hand panel shows the fibroblastoid cells which have grownfrom the Ep-Ras tumour, whereas the right-hand panel shows theepitheloid cells which have grown from the Ep-Ras-TβRII-dn tumour.

Example 11

Expression of TβRII-dn in Fibroblastoid, Highly Metastasising ColonCarcinoma Cells (CT26): Inhibition of the Invasive Growth of These Cellsin vitro, Delaying of Tumour Formation and Inhibition of the Formationof Lung Metastases in Mice

Once it was shown that the dominant-negative TGFβ receptor (TβRII-dn)could prevent both the EF conversion of EpRas cells and alsodramatically slowed down the tumour growth of these cells, it was usefulto examine the efficacy of this TβRII-dn in tumour cells which hadalready stably undergone EF conversion and were already highlymetastatic. The mouse colon carcinoma cell line CT26, an establishedmouse model for lung metastasis formation from a primary tumour(Brattain et al. 1980), was chosen. These cells were infected with anTβRII-dn-expressing retrovirus (cf. Example 10), TβRII-dn-expressingclones were selected and various clones were subjected to analysis invitro and in vivo.

Two types of TβRII-dn-expressing CT-26 clones (CT26−TβRII-dn) wereobtained. The first type showed a distinctly epithelial, but stillabnormal morphology on plastic and expressed small amounts of theepithelial markers E-cadherin and ZO-1. The second type of clone, on theother hand, on plastic formed lawns of cells with epithelial morphologywhich even formed hemicysts (domes). As expected, this second type ofclone showed high lateral expression of the epithelial markersE-cadherin and ZO-1. Control CT26 cells which were infected with aretrovirus without an insert exhibited the expected fibroblastoidmorphology on plastic and no expression of epithelial markers. It wasthus shown that TβRII-dn is capable of converting the fibroblastoid CT26cells into epithelial cells in vitro and thus effecting FE conversion.

Next, representative CT26−TβRII-dn clones of both types as well as CT26control cells are sown in collagen gels with 5% FCS. FIG. 16 shows thatthe control cells grow into the expected enormous strings and networksof fibroblastoid cells (FIG. 15, top half of the picture, left-handpanels). By contrast the CT26−TβRII-dn clones of type 1 formed compactclumps with only a few single cells growing out (FIG. 15, top half ofthe picture, middle panels) whereas the CT26−TβRII-dn clones of type 2only grew into tiny, compact groups of cells (FIG. 15, top half of thepicture, right-hand panels). This showed that TβRII-dn prevents theinvasive growth of CT26 cells in the collagen gel.

Then the same cell types were tested by the chicken heart invasion assay(cf. Example 4, FIG. 5). Whereas control-CT26 cells, as expected, grewvery invasively in this assay (FIG. 16, bottom half of the picture,left-hand panel), the CT26−TβRII-dn clones of types 1 and 2 in this testwere only slightly invasive or not invasive at all (FIG. 15, bottom halfof the picture, middle and right-hand Table.)

These experiments show that TβRII-dn causes an FE conversion of CT26cells and totally inhibits their invasive growth in two assay systems.It was therefore of great interest to examine the behaviour of thesecells in the animal. Therefore, CT26 control cells as well as 6CT26−TβRII-dn-clones of types 1 and 2 were injected into nude mice.Whereas the control animals had to be killed after 2-3 weeks on accountof their excessively large tumours, the tumour growth in mice wasdelayed with CT26−TβRII-dn-clones of type 1 by about 3-4 weeks, whereasin mice with CT26−TβRII-dn-clones of type 2 it was delayed by 6-10 weeksor totally inhibited for 24 weeks (end of experiment) (3 animals, datanot shown in the FIG.). These results show that TβRII-dn can alsodramatically delay the growth of CT26-primary tumours in some cases.

Next, the ability of the CT26−TβRII-dn cells to colonise the lung from aprimary tumour and form metastases was examined. As shown in FIG. 17(diagram in bottom half), mice were injected with CT26 control cells (3mice) or 7 different CT26−TβRII-dn-clones (type 1 and type 2, 3 mice perclone) and the growth of palpable tumours was awaited. After a certaintumour volume (4 cm³) had been reached the primary tumour was excised sothat no tumour cells remained at the injection site. The mice thustreated were examined for lung metastases after their death.

All control animals (3 mice) bearing CT26-tumours died after 2-4 weeksof lung metastases (FIG. 16, diagram in top half of picture, dottedline). By contrast the formation of lung metastases could not bedetected in any of the animals injected with CT26−TβRII-dn-clones evenafter 18 weeks (FIG. 16, diagram in top half of picture, black lines). 5animals in which there was a local recurrence of the primary tumour werenot included in the evaluation.

These data clearly show that TβRII-dn fully inhibits the metastasisationof CT26-primary tumours. Finally, the stage of metastasisation which isinhibited by TβRII-dn was checked. It is possible that only themigration of the CT26 cells out of the primary tumour into the bloodvessels is inhibited. However, the settling of the cells out of thecirculation and in the lungs might also be affected. The latter isimportant because, for example, more tumour cells might enter thecirculation of a person when a tumour was surgically removed. In orderto test this, different quantities of CT26 control cells and a number ofCT26−TβRII-dn clones of type 2 were injected intraveously into mice (3animals per cell type). The animals were then examined for lungmetastases after death. Preliminary tests showed that a mere 500 CT26cells per animal are sufficient to form lung metastases in this way.Therefore 10 and 100 times the quantity of both cell types wereinjected. FIG. 17 shows that after 14 days (50,000 cells) and 28 days(5,000 cells) the CT26 control cells had formed lung metastases in allthe animals. By contrast, even after 40 days, all the animals injectedwith CT26−TβRII-dn clones were still alive and had not yet formed anylung metastases, as confirmed on individual mice killed at this stage.Thus TβRII-dn can also prevent CT26 cells already in the circulationfrom settling in the lungs.

FIG. 15 shows that TβRII-dn inhibits both the invasive growth of CT26cells in the collagen gel, and also suppresses the invasivity of thesame cells in the chicken heart invasivity test.

For the first test (collagen gel assay, top half of the picture) CT26control cells (CT26, left-hand panels) and a CT26−TβRII-dn clone of typeI (middle panels) and type 2 (right-hand panels) were sown in collagengels with 5% serum and after 10 days microphotographs of the collagengels were prepared. The gels were photographed at two differentmagnifications (lower magnification, top panels, higher magnification,lower panels). Whereas the CT26 control cells grew into largenetwork-shaped and string-like structures, consisting of spindle-shaped,fibroblastoid cells (left-hand panels), the CT26−TβRII-dn type 1 cellsformed compact clumps of cells with very few cells growing into the gel(middle panels). The CT26−TβRII-dn clones of type 2 form only tinycompact cell groups without any ability to grow into the collagen gel(right-hand panels).

For the chicken heart invasion assay (bottom half of the picture) thetest cells were charged with a fluorescent vital dye, brought intocontact with chicken heart fragments and examined histologically after 7days (cf. methods and Example 4). The control cells migrated efficientlyinto the chicken heart fragment (left-hand Table, light-coloured groupsof cells and strings on the side of the boundary between test cells andchicken heart fragment indicated by a dotted line labelled H). Bycontrast type 1 clones migrated only slightly into the chicken hearttissue (middle panel, cf. the few light-coloured cells in the areamarked H), while the CT26−TβRII-dn cells of type 2 did not growinvasively at all (all the light-coloured cells remained outside thechicken heart fragments H (dotted line).

FIG. 16 shows that the expression of TβRII-dn in CT26 cells blocks theirability to form lung metastases from a primary tumour.

The progress of the experiments is shown diagrammatically in the bottomhalf of the picture. 7 different CT26−TβRII-dn clones (type 1 and 2,CT26+TβRII-dn) as well as CT26 control cells were used in the test.Syngenic Balb-C mice (3 per cell type) were injected with 1×10⁶ cellsper animal and the growth of tumours was awaited. After the primarytumours reached a size of 4 cm³ they were surgically removed and afterthey had died the mice were examined for lung metastases. The resultsare shown in the diagram (top half of the picture). Whereas the 3control animals died within 4 weeks of lung metastases (dotted line),all the animals treated with CT26−TβRII-dn cells were still alive andfree from lung metastases after 18 months (black lines). In the case ofthe line ending after 14 weeks (top diagram) the primary tumour reachedthe critical size so late that 18 weeks had not passed by the time thetest ended.

FIG. 18 shows that TβRII-dn in CT26 cells also inhibits their ability tosettle in the lungs from the bloodstream and form metastases there.

The diagram in the top part of the Figure shows the progress of theexperiment. Syngenic Balb-C mice (3 per cell type and cell quantity)were injected intravenously (into the caudal vein) with CT26 controlcells and several CT26−TβRII-dn clones. The Figure shows that all threemice treated with 5,000 or 50,000 control cells (CT26) had died of lungmetastases after 28 or 14 days (+), whereas all the animals injectedwith CT26−TβRII-dn clones were still alive free from lung metastasesafter 40 days (−)

Example 12

The activated TGFβ receptor activates the transient transcription of aPAI-1-promoter-reporter gene construct, a process which is inhibited byTβRII-dn.

With a view to discoveing TGFβ-(receptor) inhibitors by means ofcellular assay in a High Throughput screening (HTS) process a test cellis prepared as follows: a PAI-1-promoter-reporter gene construct isstably expressed in a suitable cell (Ep-Ras or CT26). At the same timethe human TGFβ receptor chain (e.g. TβRII) selected for the screening isexpressed in this cell. By contrast, an unrelated receptor which alsoinduces PAI-1 transcription, e.g. an FGF receptor, is expressed incontrol cells in addition to the PAI-1-reporter construct.

A prerequisite for the development of a test cell line of this kind isthat the TGFβ-induced PAI-I expression (suppressed by the inhibitor) iscorrelated with the tumour formation or metastasisation of thecorresponding cells in transient transfection tests. In order to checkthis, CT26 control cells and 5 CT26−TβRII-dn clones already tested inmice (cf. Example 11, FIG. 16) were transfected with the 3TP-luxPAI-1-reporter gene construct (Wrana et al., 1992), stimulated with TGFβor left untreated and tested for PAI-1 expression (measurement ofluciferase activity). As positive and negative controls a constitutivelyactive TGFβR chain (TβRI(T204D); Wrana et al. 1994) as well as TβRII-dnDNA together with the PAI-1 reporter construct were co-transfected intountreated CT26 cells. FIG. 18 shows that untreated CT26 cells (CT26controls) without TGFβ treatment have a basal activity which correspondsto that of the negative controls (cotransfection of TβRII-dn). In thesame control cells TGFβ activates the reporter gene transcription tolevels which are attained in the positive controls by cotransfection ofa constitutively active TGFβ receptor.

Different TβRII-dn expressing CT26 clones behaved differently in thistest (FIG. 18, CT26−TβRII-dn 1-5). In two clones (CT26−TβRII-dn 3 and 4)the luciferase activity achieved after TGFβ stimulation was below or atthe levels found in the negative controls, irrespective of whether thecells were tested before injection or after isolation from the veryslow-growing tumour. In the three remaining clones (in which the tumoursinduced grew faster) the luciferase activity was at the level of thenegative controls only before injection into the animal, after isolationfrom the tumour intermediate levels or even activities comparable tothose in the positive controls were found (FIG. 18). It can be assumedthat in the latter clones there was selection for cells in which theexpression of TβRII-dn was down-regulated. This assumption was supportedby the fact that renewed selection of the cells from the tumour inpuromycin again killed off many cells and the survivingpuromycin-resistant cells no longer exhibited increased PAI-1transcription after TGFβ stimulation. The results of these experimentsshow that the tumour formation/metastasisation in the animal is clearlycorrelated with the TGFβ-activatability of a PAI-1 promoter-reportergene construct.

FIG. 18 shows that the expression of TβRII-dn in CT26 cells suppressesthe TGFβ-induced transcription of a PAI-1 promoter-reporter geneconstruct.

CT26-control cells (CT26 controls) and 5 clones of CT26−TβRII-dn cells(CT26TβRII-dn 1-5) were transfected with a PAI-1 promoter-reporter geneconstruct (3TP-lux), the cells were stimulated with TGFβ (+TGFβ) or leftunstimulated (−TGFβ) and the luciferase activity was measured in cellextracts. As positive controls the cDNA of a constitutively active TGFβreceptor chain 1 (TβRI(T204D; Wrana et al., 1994) as well as theTβRII-dn-cDNA together with 3TP-lux were cotransfected into the cells.This measurement was carried out in cells before injection into theanimal (before tumour induction) and after isolation and cultivation ofthe tumour cells for 3 days (isolated tumour cells) (cf. legend, box attop right). The bars indicate the standardised luciferase activity fromextracts with the same protein content (cf. methods).

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What is claimed is:
 1. A process for screening a test substance toidentify the presence therein of a pharmacologically active substance,useful for the treatment of epithelial, invasive tumour disease,comprising: contacting a mammalian cell with said test substance, anddetermining whether the signal transduction pathway initiated by TGFβ insaid mammalian cell is inhibited, wherein said pharmacologically activesubstance is detected by the inhibition of said signal transductionpathway and said pharmacologically active substance is not TGFβ,anti-TGFβ antibody or antisense TGFβ RNA.
 2. The process according toclaim 1, wherein said pharmacologically active substance is ananti-tumour agent, wherein said anti-tumour agent inhibits the growth ofepithelial invasive tumour disease cells.
 3. The process according toclaim 2, wherein said epithelial invasive tumour disease cells arecharacterized by a reversible transition of the cells from anepithelial, non-invasive state into an invasive state.
 4. The processaccording to claim 1, wherein said mammalian cell is transformed with(a) a plasmid containing a reporter gene which is under the control ofthe regulatory sequence of a cell protein regulated by TGFβ; or (b) aplasmid containing a DNA sequence coding for a functional mammalian TGFβreceptor.
 5. The process according to claim 4, wherein said mammaliancell is grown in culture.
 6. The process according to claim 4, whereinsaid mammalian cell is a human cell.
 7. The process according to claim4, wherein said cell is transformed with a plasmid containing a DNAsequence coding for TGFβ receptor type II.
 8. The process according toclaim 4, wherein said reporter gene is under the control of theregulatory sequence of the plasminogen activator inhibitor.
 9. Theprocess according to claim 4, wherein said inhibition is determined bymeasuring the modulation, by said test substance, of theautophosphorylation of (a) the TGFβ receptor type II or (b) thecytoplasmic domain of said TGFβ receptor type II.
 10. The processaccording to claim 4, wherein said inhibition is determined by measuringthe modulation, by said test substance, of the ability of the TGFβreceptor type II to phosphorylate the TGFβ receptor type I or its GSdomain.
 11. The process according to claim 1, wherein said mammaliancell is a human cell.
 12. The process according to claim 1, comprising:measuring the rate of TGFβ signal transduction in a first mammalian cellcomprising a functional TGFβ receptor wherein said receptor is activatedby the addition or presence of a selected concentration of TGFβ;contacting a second mammalian cell comprising said functional TGFβreceptor with said test substance; activating the functional TGFβreceptor in said second mammalian cell by the addition or presence ofsaid selected concentration of TGFβ; measuring the rate of TFGβ signaltransduction in said second mammalian cell; wherein the presence of saidpharmacologically active substance in said test substance is detectedwhen the measured rate of TGFβ signal transduction in said secondmammalian cell is less than the measured rate of TGFβ signaltransduction in said first mammalian cell.
 13. The process according toclaim 12, wherein said first mammalian cell and said second mammaliancell are transformed with (a) a plasmid containing a reporter gene whichis under the control of the regulatory sequence of a cell proteinregulated by TGFβ; or (b) a plasmid containing a DNA sequence coding fora functional mammalian TGFβ receptor.
 14. A process for screening a testsubstance to identify the presence therein of a pharmacologically activesubstance, useful for the treatment of epithelial, invasive tumourdisease, comprising: contacting a test substance with a mammalian cell,wherein said mammalian cell is transformed with (a) a plasmid containinga reporter gene which is under the control of the regulatory sequence ofa cell protein regulated by TGFβ, or (b) a plasmid containing a DNAsequence coding for a functional mammalian TGFβ receptor; anddetermining whether the signal transduction pathway initiated by TGFβ insaid mammalian cell is inhibited; wherein said pharmacologically activesubstance is detected by the inhibition of said signal transductionpathway and said pharmacologically active substance is not TGFβ,anti-TGFβ antibody or antisense TGFβ RNA.